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Dec 2018

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Assessing Olfaction Using Ultrasonic Vocalization Recordings in Mouse Pups with a Sono-olfactometer
使用超声波嗅觉计通过超声波发生记录评估小鼠幼崽的嗅觉   

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Abstract

Olfaction is the first sensory modality to develop during fetal life in mammals, and plays a key role in the various behaviors of neonates such as feeding and social interaction. Odorant cues (i.e., mother or predator scents) can trigger potentiation or inhibition of ultrasonic vocalizations (USV) emitted by pups following their isolation. Here, we report how USV are inhibited by olfactory cues using a sono-olfactometer that has been designed to quantify precisely olfaction in pups congenitally infected by cytomegalovirus. This olfactory-driven behavioral test assesses the USV emitted in presence of unfamiliar odorants such as citral scent or adult male mouse scent. We measure the number of USV emitted as an index of odorant detection during the three periods of the 5-min isolation time of the pup into the sono-olfactometer: first period without any odorant, second period with odorant exposure and last period with exhaust odorant. This protocol can be easily used to reveal olfactory deficits in pups with altered olfactory system due to toxic lesions or infectious diseases.

Keywords: Olfactory signals (嗅觉信号), Odor detection (气味检测), Ultrasonic call (超声波电话), Behavioral inhibition (行为抑制), Pup development (小狗发育), Fear (恐惧), Isolation (隔离), Congenital cytomegalovirus infection (先天性巨细胞病毒感染)

Background

In mammals, olfaction is the first sensory sense to become functional in utero, long before audition and vision (Stickrod et al., 1982; Sarnat and Yu, 2016). Survival and growth of neonates depend on the mother and rely heavily on their reciprocal olfactory-driven behaviors such as nipple localization, feeding, attachment, predator avoidance, etc. (Teicher and Blass, 1976; Brunjes and Alberts, 1979; Bell and Smotherman, 1980; Brouette-Lahlou et al., 1992; Shair et al., 1999; Hongo et al., 2000; Perry et al., 2016; Al Aïn et al., 2017). Thus, congenital or perinatal impairment of the sense of smell, such as toxic or infectious injury of the olfactory system, could have profound health concerns. While a great number of non-operant and operant behavioral tests are available for assessing the sense of smell in adult rodents (Bodyak and Slotnick, 1989; Slotnick and Restrepo, 2005; Kobayakawa et al., 2007; Yang and Crawley, 2009), there is a serious limitation in exploring olfaction in very young rodents due to their limited behavioral repertoire. Nevertheless, a suitable test to address odor perception in rodent pups has been designed based on the recording of ultrasonic isolation calls (Hofer and Shair, 1991; Hofer et al., 2002; Lemasson et al., 2005; Lazarini et al., 2018). Young pups, while isolated from the mother and littermates and placed in low ambient temperature produce ultrasonic vocalizations (USV) at a high rate (Smith and Sales, 1980; Branchi et al., 1998; Castellucci et al., 2018), that promote maternal behavior such as searching for pups, retrieving and licking of pups (Noirot, 1974; Brounette-Lahlou et al., 1992; Brunelli et al., 1994). Infants of most mammals, including humans, also emit repeated vocalizations in the audible range after isolation, as a distress signal aiming at eliciting maternal behavior. USV emission of the isolated rodent pups stops at the contact of the mother, littermates or nest odor (Szentgyörgyi et al., 2008). On the one hand, potentiation of the USV response to isolation can be induced by exposition to the scent of its mother or another lactating female (Shair et al., 1999). On the other hand, inhibition of the ultrasonic calls could be induced by exposition to the scent of an unfamiliar adult male, one of its common predators in the wild (Shair et al., 1999). USV inhibition can also be achieved by exposure to a non-social odorant cue, such as citral, that triggers innate aversive response (Lemasson et al., 2005). Using this olfactory-induced USV inhibition, we found that congenital cytomegalovirus (CMV) infection alters olfaction as early as Day 6 after birth, long before hearing deterioration in mice (Lazarini et al., 2018).

The current protocol describes a method for assessing odor perception in young mouse pups soon after birth, using a custom-made sono-olfactometer. Because this approach has been developed in the context of congenital viral infection, the sono-olfactometer was designed to prevent any spread of the virus from infected pups to the environment. It can be used therefore in Biosafety level 1 (BSL-1), level 2 (BSL-2) or level 3 (BSL-3) according to the microbial status of the manipulated animals. This characteristic was made possible by slightly modifying the earlier version of our olfactometers (Lemasson et al., 2005). The pup chamber of the sono-olfactometer constitutes a mini-isolator in which odorants can be presented at a constant concentration and then efficiently exhausted. This sono-olfactometer allows simultaneous exposition to various odorants and recordings of USV emitted by the pup placed in the chamber. This protocol can be easily expanded to explore olfaction in other paradigms of acute and chronic injury or infectious diseases in the olfactory system of wild-type or genetically-modified rodents.

Materials and Reagents

  1. 50-ml tube (Corning, France, catalog number: 430828) with two custom-made 5 mm-diameter holes in the lid
  2. Laboratory-bred mouse pups from 6-8 days after birth
    Notes: 
    1. Put in a single cage each pregnant female one week before the timed day of birth. 
    2. For the test, male and female pups can be used. We only tested Oncins France 1 (OF1) mouse line from Charles Rivers, France with this behavioral protocol. This mouse strain is productive and widely used for teratology. Pups of other mouse strains such as C57Bl/6J emit similar USV (Castellucci et al., 2018).
    3. Infected-pups can be used as previously described (Lazarini et al., 2018). OF1 mother mice and its litter from Charles Rivers, France were individually housed in two isolators, one for the control (CTL) group and the second for the CMV group, kept in a BSL-2 room with controlled temperature (22 °C) and humidity (range: 40%-70%), under 12 h light/dark cycle (lights on at 8:00 AM) in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents. Mice were manipulated in class II safety cabinets.
    4. You can identify the pups at Day 1 after birth using long-lasting paw tattoos, subcutaneously injected with a 0.3 mm x 13 mm needle.
  3. Male scent (10 g soiled bedding from a group of 6 unfamiliar male adult OF1 mice)
  4. Mineral oil (Sigma, France catalog number: M5904) 
  5. Citral (Sigma, France catalog number: W230316)
    Note: Citral has lemon scent.
  6. 70% Ethanol solution
  7. Citral solution (see Recipes)

Equipment

  1. Class II safety cabinets
  2. Custom-made sono-olfactometer (depicted in Figure 1)
    1. The audio recording system is composed of:
      1. A sound card recorder (PreSonus AudioBox iTwo) (Figure 1). The sound card can be replaced by any other commercial model. The recording potentiometer is adjusted to maximize the signal-to-noise ratio and avoid overloading.
      2. A heterodyne bat detector (whose microphone has been moved into the chamber isolator using a BNC cable) (Magenta Bat5 Digital Bat Detector, RSPB, UK) (Figures 1 and 2). The heterodyne bat detector is set to the center frequency of the mouse vocalization: The volume control of the bat detector is adjusted in the middle to avoid background noise.


      3. Figure 1. The sono-olfactometer. The sono-olfactometer is a system that delivers odors while recording the ultrasound emitted by mice. It is composed of four sub-systems: 1) An audio recording system; 2) Two identical isolated chambers (only one chamber will be presented); 3) An odor dispenser (olfactometer); 4) A computer with its own software.


    2. The isolated chamber (Figure 2):
      1. Exhaust air pump (Schego, catalog number: 850)
      2. Non-return (check) valve (composed of two elements from Colder Products, catalog numbers: PLCD220-04 and PLCD10004)
      3. Exhaust air flow meter (Brooks Instrument, catalog number: FR2A13BVBN)


      Figure 2. The isolated chamber. The isolated chamber (inside size 18 x 12 x 12 cm) is sealed and airtight when closed. The isolated chamber was made with components (8 mm-thick black PVC walls for the ceiling, the floor and sides, 8 mm-thick transparent plexiglass for the door, 5 mm-diameter rubber seal for the door, 0.1 mm-thick aluminum coating plate with 2 mm diameter holes) easily available in DIY stores such as Lacrylic shop, Bonneuil sur Marne, France. The odor port and the ultrasonic microphone are on the right side of the chamber. The odor dispenser releases the Odor/Air mixture in a controlled manner through the odor port (Figure 3). A drain hole in the center of the left side is connected to a HEPA exhaust air filter (Millex-FG, 0.20 µm, PTFE hydrophobe, 50 mm). The air is evacuated by an exhaust air pump, a non-return (check) valve (composed of two elements from Colder Products, and an exhaust air flow meter (Figure 3), thus avoiding microbial contamination of the environment. The exhaust air containing odorants is directly diverted to the air exhaust of the animal facility. The HEPA exhaust air filter is changed at the end of the experiment, after the testing of all the pups. The ultrasonic microphone is offset inside the box.

    3. The odor dispenser (olfactometer) (Figure 3):
      1. Emitting air pump (Schego, catalog number: M2K3)
      2. Air flow meter (Key Instrument, catalog number: FR2A14BVBN)
      3. Odor flow meter (Brooks Instrument, catalog number: FR2A13BVBN)
      4. Odor valves (e.g., ASCO, catalog number: SCH284A005.12/DC or Bio-Chem Fluidics, catalog number: 100P2NC12-05B or equivalent normally closed solenoid pinch valves)
      5. Exhaust air pump (Schego, catalog number: 850)
      6. C-Flex® Standard Tubing (ID: 0.125 OD: 0.250)
        Note: The tubing is GMP Compliant.
  3. Note: All electronics used to control the system is a custom-made device but it could easily be replaced by a commercialized version of Arduino card: https://www.arduino.cc/en/Main/Products.


    Figure 3. The odor dispenser. The odor dispenser distributes the Odor/Air mixture in a controlled manner. The air from the emitting air pump is transferred to the air flow meter and the odor flow meter. This device allows one to adjust the Odor/Air ratio (0.3 L/min for odorant and 2 L/min for air). If odor valves (can be “ASCO”, “Bio-Chem Fluidics” or equivalent normally closed solenoid pinch valves) are closed, then clean air will be diffused into the chamber, otherwise the odor will be diffused homogeneously in the chamber. The air is evacuated into the isolated chamber by the exhaust air pump at a flow rate of 1.5 L/min. C-Flex® Standard Tubing (ID: 0.125 OD: 0.250) is used to connect the different devices.

  4. Personal protective equipment (PPE)
    May include (but is not limited to) scrubs, a sterile combination, latex gloves, bouffant cap, ventilation mask, protective glasses, and shoe covers, depending on the regulation of the animal facility in which the work is taking place.

Software

  1. The software (Figure 4):
    Pups emitted ultrasonic vocalizations at 40-120 kHz that were detected using an ultrasonic microphone connected to a bat detector (frequency range 10-130 kHz) that converts ultrasonic sounds to the audible frequency range. Using the broadband 60 kHz output of the detector, ultrasonic calls were sampled, recorded and analyzed using Audacity open software (www.audacityteam.org).


    Figure 4. The audio recording software. This figure shows the computer screen with the open images of the audio recording and the odor diffusion software. The control of the diffusion of odors is ensured by a custom-made software. This software can be replaced by the Node-RED software coupled with an Arduino. As the Arduino appears as a Serial device, the Serial in/out nodes can be used to communicate with it (https://nodered.org/docs/hardware/arduino).

  2. GraphPad Prism software (GraphPad Software, USA) is used for data analysis

Procedure

Notes:

  1. Gloves should be worn for all steps that involve handling mice, odorants, the sono-olfactometer and its chambers. 
  2. Prior to bringing the pups into the sono-olfactometer, prepare the odorants, the chambers (ensure cleanliness, connectivity of wires and odor dispensers, the bat detector and the computer software.
  3. Prepare a sheet with all the animal information including animal numbers, feet marking, genotype, treatment and/or inoculum, chamber number, weight, etc.
  4. Five minutes prior to testing, pups are moved from the homeroom and eventually from colony isolator to the class II safety cabinets in the testing room, in their home cages with their dams and litter. Two pups were placed into the two detachable chambers of the sono-olfactometer under the class II safety cabinets (one pup per chamber); the chambers with the pups were then put into the laboratory-made sono-olfactometer placed in the BSL-2 room, at proximity to the class II safety cabinets, for USV-recordings.
  5. Each test session in the sono-olfactometer lasts 5 min. Pups can be tested two by two, on Days 6 and 8 after birth.
  6. There is a maximum of one session per day, with each session comprised of exposure to only one scent.
  7. Pups should be weighed after the test every day (balance placed in the safety cabinet) prior to be placed back to the nest. They should be separated from their mother less than 30 min in total (time including transfer to the chamber, testing, weighing and transfer to the home cage).

Recording of USV (Figure 5)


Figure 5. Recordings and quantification of the emission of ultrasonic vocalizations. A and B. The recording of ultrasonic calls began 30 s after placing the pups in the test chamber of the sono-olfactometer. Ultrasonic vocalizations were detected using an ultrasonic microphone connected to a bat detector that converts ultrasonic sounds into the audible frequency range (from 20 to 20,000 Hz). C. Experimental paradigm. Ultrasonic emissions 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 air (1 min and 30 s. This time duration allows the complete elimination of the exhaust air containing odorants). D. Typical wave traces of spontaneous call series from a pre-weaning 6-day-old pup (for more details, see Lazarini et al., 2018). The majority of vocalizations (vocal units of duration < 100 ms on spectrogram) are produced in series with call intervals > 130 ms.


  1. Place the two chambers containing the pups onto the table of the sono-olfactometer. Connect the sono-olfactometer (SO) and the computer. Switch on the bat detector.
  2. Thirty seconds after connecting the two chambers to the sono-olfactometer, record the USV by starting the audio recording software. Record simultaneously the USV emitted by the two pups, each in their own sealed chamber. The routine protocol is shown in Figure 6. An example of USV recording is Sound 1 (this audio file depicts the USV of two 2-day-old pups).


    Figure 6. USV recording in a sono-olfactometer. The successive stages of the experimental procedure. Using the broadband 60 kHz output of the detector, ultrasonic calls were sampled, recorded and analyzed using the Audacity open software.

  3. Sixty seconds after the beginning of USV recording, start the odor diffusion software. 
  4. When the odor diffusion program is finished (duration: 5 min), include the date, experiment number and all other needed information in the file name.
  5. Transport the pups to their home cage (transfer from the chambers to the safety cabinet, weight them and transfer to the nest of the home cage). 
  6. Clean the chambers using 70% ethanol. After a 5-min interval to allow the elimination of the ethanol odorant, start the next test using the same chambers with other pups. 
  7. The USV records can now be analyzed using the Audacity software. An example of analysis is displayed in Figure 7.


    Figure 7. Sample data of the olfactory USV inhibition test. A. Timetable of the experiments. All pups in the same litter of timed pregnant mice were individually infected in utero at embryonic day 13 (E13) with intraplacental inoculation of murine CMV (Smith strain) under anesthesia. As a control (CTL) group, all pups in the same litter of other timed pregnant mice were intraplacentally injected with PBS at E13 under anesthesia. Animals were analyzed using sono-olfactometers on Days 6 and 8 after birth. B and C. Emission of ultrasonic calls for citral odorant on Day 6 after birth (n = 18 CTL, n = 19 CMV). D and E. Emission of ultrasonic calls for male scent odorant on Day 8 after birth (n = 8 CTL, n = 11 CMV). P values are calculated by Wilcoxon matched-pairs signed rank test. **P < 0.01, ****P < 0.0001; mean ± SEM in B-E. CTL pups decrease their emission of calls in response to exposure to non-social or social odorant molecules, such as citral or male scent, respectively. In contrast, congenital CMV infection impairs the ultrasonic call responses triggered by the two scents, indicating an alteration of olfactory perception induced by the virus (for more details, see Lazarini et al., 2018).

Data analysis

The number of ultrasonic vocalizations emitted after isolation was manually counted using Audacity open software (www.audacityteam.org). The mean rate of ultrasonic emissions (call/min) was computed for each time block: The first period without any odorant (1 min), the second period with exposure to social or non-social odorant (1 min) and the last period with exhaust odorant (1 min and 30 s). Data were analyzed using GraphPad Prism, using Mann-Whitney or Wilcoxon matched-pairs signed rank tests as appropriate.

Notes

  1. Prior to testing pups, we recommend validating the two sono-olfactometer chambers by recording successively in each of them the same CTL pup aged of 2 days.
  2. Since this test is a non-operant one (recording of spontaneous USV emitted by pups exposed to different olfactory cues), at least 8 pups per group should be used to reach the statistical power necessary for the analysis. 
  3. When manipulating two groups of animals, one infected and the other non-infected, we recommend dedicating each of the two SO chambers to each group using an external labeling. This should help in preventing any contamination of CTL pups.
  4. The dilutions of Citral odorant should be made just before testing. We recommend preparing the scent under a fume hood in a laboratory outside the animal facilities since it is important to prevent the odor diffusion in the animal facilities (odor habituation).
  5. For a greater reproducibility, fresh male bedding should be used before any microbial transformation of the scents.

Recipes

  1. Citral solution
    1 ml Citral
    ad 10 ml mineral oil dilution

Acknowledgments

The predecessor to the presented sono-olfactometer (i.e., the murine USV inhibition task based on an eight-channel olfactometer as described in Slotnick and Bodyak, 1999), was co-developed by Drs Morgane Lemasson, Gilles Gheusi and Pierre-Marie Lledo (first published as Lemasson et al., 2005). The funding for the development of the sono-olfactometers was provided in a grant of the Institut Pasteur, Paris (GPF 2015 Microbes & brain “INFECSMELL”) awarded to FL.

Competing interests

These results are the subject-matter of a U.S. provisional patent application number 62/793941 filed on 18 January 2019 on which Françoise Lazarini, Sébastien Wagner and Pierre-Marie Lledo are cited as inventors.

Ethics

All animal procedures were performed in accordance to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013-118, February 6th, 2013), according to the regulations of Inserm and Pasteur Institute Animal Care Committees. The Animal Experimentation Ethics Committee (CETEA 89) of the Pasteur Institute has approved this study (2015-0028). Mice were housed in isolators and manipulated in class II safety cabinets in the Pasteur Institute animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents.

References

  1. Al Aïn, S., Perry, R. E., Nunez, B., Kayser, K., Hochman, C., Brehman, E., LaComb, M., Wilson, D. A. and Sullivan, R. M. (2017). Neurobehavioral assessment of maternal odor in developing rat pups: implications for social buffering. Soc Neurosci 12(1): 32-49.
  2. Bell, R. W., Smotherman, W. P. (1980). Maternal Influences and Early Behavior. Spectrum Publ. Inc., London, pp. 105-133.
  3. Bodyak, N. and Slotnick, B. (1999). Performance of mice in an automated olfactometer: odor detection, discrimination and odor memory. Chem Senses 24(6): 637-645.
  4. Branchi, I., Santucci, D., Vitale, A. and Alleva, E. (1998). Ultrasonic vocalizations by infant laboratory mice: a preliminary spectrographic characterization under different conditions. Dev Psychobiol 33(3): 249-256.
  5. Brouette-Lahlou, I., Vernet-Maury, E. and Vigouroux, M. (1992). Role of pups' ultrasonic calls in a particular maternal behavior in Wistar rat: pups' anogenital licking. Behav Brain Res 50(1-2): 147-154.
  6. Brunelli, S. A., Shair, H. N. and Hofer, M. A. (1994). Hypothermic vocalizations of rat pups (Rattus norvegicus) elicit and direct maternal search behavior. J Comp Psychol 108(3): 298-303.
  7. Brunjes, P. C. and Alberts, J. R. (1979). Olfactory stimulation induces filial preferences for huddling in rat pups. J Comp Physiol Psychol 93(3): 548-555.
  8. Castellucci, G. A., Calbick, D. and McCormick, D. (2018). The temporal organization of mouse ultrasonic vocalizations. PLoS One 13(10): e0199929.
  9. Hofer, M. A. and Shair, H. N. (1991). Trigeminal and olfactory pathways mediating isolation distress and companion comfort responses in rat pups. Behav Neurosci 105(5): 699-706.
  10. Hofer, M. A., Shair, H. N. and Brunelli, S. A. (2002). Ultrasonic vocalizations in rat and mouse pups. Curr Protoc Neurosci Chapter 8: Unit 8.14.
  11. Hongo, T., Hakuba, A., Shiota, K. and Naruse, I. (2000). Suckling dysfunction caused by defects in the olfactory system in genetic arhinencephaly mice. Biol Neonate 78(4): 293-299.
  12. Kobayakawa, K., Kobayakawa, R., Matsumoto, H., Oka, Y., Imai, T., Ikawa, M., Okabe, M., Ikeda, T., Itohara, S., Kikusui, T., Mori, K. and Sakano, H. (2007). Innate versus learned odour processing in the mouse olfactory bulb. Nature 450(7169): 503-508.
  13. Lazarini, F., Katsimpardi, L., Levivien, S., Wagner, S., Gressens, P., Teissier, N. and Lledo, P. M. (2018). Congenital cytomegalovirus infection alters olfaction prior to hearing deterioration in mice. J Neurosci 38(49): 10424-10437.
  14. Lemasson, M., Delbé, C., Gheusi, G., Vincent, J. D. and Lledo, P. M. (2005). Use of ultrasonic vocalizations to assess olfactory detection in mouse pups treated with 3-methylindole. Behav Processes 68:13-23.
  15. Noirot, E. (1974). Nest-building by virgin female mouse exposed to ultrasound from inaccessible pups. Anim Behav 22: 410-420.
  16. Perry, R. E., Al Ain, S., Raineki, C., Sullivan, R. M. and Wilson, D. A. (2016). Development of odor hedonics: experience-dependent ontogeny of circuits supporting maternal and predator odor responses in rats. J Neurosci 36(25): 6634-6650.
  17. Sarnat, H. B. and Yu, W. (2016). Maturation and dysgenesis of the human olfactory bulb. Brain Pathol 26(3): 301-318.
  18. Shair, H. N., Masmela, J. R. and Hofer, M. A. (1999). The influence of olfaction on potentiation and inhibition of ultrasonic vocalization of rat pups. Physiol Behav 65(4-5): 769-772.
  19. Slotnick, B. and Restrepo, D. (2005). Olfactometry with mice. Curr Protoc Neurosci Chapter 8: Unit 8.20. 
  20. Smith, J. C., and Sales, G. D. (1980). Ultrasonic behavior and mother-infant interactions in rodents. In: Maternal Influences and Early Behavior. Bell, R. W. and Smotherman, W. P. (Eds.). SP Medical and Scientific Books, New York, pp. 105-133.
  21. Stickrod, G., Kimble, D. P. and Smotherman, W. P. (1982). In utero taste/odor aversion conditioning in the rat. Physiol Behav 28(1): 5-7.
  22. Szentgyörgyi, H., Kapusta, J. and Marchlewska-Koj, A. (2008). Ultrasonic calls of bank vole pups isolated and exposed to cold or to nest odor. Physiol Behav 93(1-2): 296-303.
  23. Teicher, M. H. and Blass, E. M. (1976). Suckling in newborn rats: eliminated by nipple lavage, reinstated by pup saliva. Science 193(4251): 422-425.
  24. Yang, M. and Crawley, J. N. (2009). Simple behavioral assessment of mouse olfaction. Curr Protoc Neurosci Chapter 8: Unit 8.24.

简介

嗅觉是哺乳动物在胎儿生命中发展的第一种感觉形态,并且在新生儿的各种行为中起着关键作用,如喂养和社交互动。气味提示(即,母亲或捕食者气味)可以触发幼犬在分离后发出的超声波发声(USV)的增强或抑制。在这里,我们报告了如何使用声嗅觉计来抑制USV的嗅觉提示,该声嗅觉计被设计用于量化先天性巨细胞病毒感染的幼崽的精确嗅觉。这种嗅觉驱动的行为测试评估了在不熟悉的气味如柠檬醛气味或成年雄性小鼠气味存在的情况下排放的USV。我们测量USV排放的数量,作为在幼崽进入声嗅觉仪的5分钟隔离时间的三个时期内的气味检测指数:第一阶段没有任何气味,第二阶段有气味暴露,最后一期有排气味。 。该方案可以很容易地用于揭示由于中毒性病变或传染病导致嗅觉系统改变的幼崽的嗅觉缺陷。
【背景】在哺乳动物中,嗅觉是第一次在试验和视觉之前成为宫内功能的感觉(Stickrod et al。,1982; Sarnat和Yu,2016)。新生儿的生存和成长取决于母亲,并严重依赖其相互嗅觉驱动的行为,如乳头定位,喂养,依恋,捕食者避免,等。 (Teicher和Blass,1976; Brunjes和Alberts,1979; Bell和Smotherman,1980; Brouette-Lahlou et al。,1992; Shair et al。,1999; Hongo et al。,2000; Perry et al。,2016;AlAïn et al。,2017)。因此,嗅觉的先天性或围产期损害,例如嗅觉系统的毒性或感染性损伤,可能具有深刻的健康问题。虽然有大量的非操作性和操作性行为测试可用于评估成年啮齿动物的嗅觉(Bodyak和Slotnick,1989; Slotnick和Restrepo,2005; Kobayakawa et al。,2007; Yang和Crawley,2009),由于行为特征有限,在非常年轻的啮齿动物中探索嗅觉存在严重的局限性。尽管如此,基于超声波隔离呼叫的记录,设计了一种解决啮齿动物幼崽气味感知的合适测试(Hofer和Shair,1991; Hofer et al。,2002; Lemasson et al 。,2005; Lazarini et al。,2018)。年幼的幼崽虽然与母亲和同窝小鼠隔离并且处于低环境温度下,但却以高比率产生超声波发声(USV)(Smith and Sales,1980; Branchi et al。,1998; Castellucci et al。,2018),促进母性行为,如寻找幼崽,检索和舔幼崽(Noirot,1974; Brounette-Lahlou et al。,1992; Brunelli et al。,1994)。大多数哺乳动物(包括人类)的婴儿在隔离后也会在可听范围内发出重复的发声,作为旨在引发母体行为的求救信号。孤立的啮齿动物幼崽的USV排放在母亲,同窝仔或巢气味的接触处停止(Szentgyörgyi et al。,2008)。一方面,USV对分离的反应的增强可以通过暴露于其母亲或另一个哺乳期女性的气味来诱导(Shair 等人,,1999)。另一方面,通过暴露于不熟悉的成年雄性动物的气味可以诱导对超声波呼叫的抑制,成年雄性是野外常见捕食者之一(Shair et al。,1999)。 USV抑制也可以通过暴露于引发先天厌恶反应的非社会气味线索(例如柠檬醛)来实现(Lemasson 等人,,2005)。使用这种嗅觉诱导的USV抑制,我们发现先天性巨细胞病毒(CMV)感染早在出生后第6天就会改变嗅觉,早在小鼠听力恶化之前很长时间(Lazarini et al。,2018)。

目前的协议描述了一种使用定制的声嗅测量仪评估出生后不久幼鼠幼崽气味感知的方法。由于这种方法是在先天性病毒感染的背景下开发的,因此声嗅测量仪旨在防止病毒从受感染的幼崽传播到环境中。因此,根据操纵动物的微生物状态,它可以用于生物安全1级(BSL-1),2级(BSL-2)或3级(BSL-3)。通过稍微修改我们的嗅觉计的早期版本可以实现这一特性(Lemasson et al。,2005)。声嗅测量计的幼崽室构成一个微型隔离器,其中气味剂可以以恒定的浓度呈现,然后有效地耗尽。这种声嗅觉仪可以同时暴露于各种气味剂和放置在腔室中的幼崽发出的USV记录。该方案可以很容易地扩展,以探索在野生型或转基因啮齿动物的嗅觉系统中的急性和慢性损伤或传染病的其他范例中的嗅觉。

关键字:嗅觉信号, 气味检测, 超声波电话, 行为抑制, 小狗发育, 恐惧, 隔离, 先天性巨细胞病毒感染

材料和试剂

  1. 50毫升管(法国康宁,目录号:430828),盖子上有两个定制的5毫米直径的孔
  2. 出生后6-8天实验室培育的小鼠幼崽
    注意:&nbsp;
    1. 在出生时间前一周,将每位孕妇放入一个笼子里。
    2. 对于测试,可以使用雄性和雌性幼仔。我们仅使用此行为方案测试了来自法国Charles Rivers的Oncins France 1(OF1)小鼠品系。这种小鼠品系具有生产力并广泛用于畸形。其他小鼠品系如C57Bl / 6J的幼崽发出类似的USV(Castellucci 等 ,2018)。
    3. 可以如前所述使用感染幼崽(Lazarini 等 ,2018)。来自法国Charles Rivers的OF1母鼠及其垫料分别饲养在两个隔离器中,一个用于对照(CTL)组,第二个用于CMV组,保持在BSL-2室中,温度控制在22℃(22℃)。在法国农业部认可的巴斯德研究所动物设施中,在12小时光照/黑暗循环(上午8:00点亮)下进行湿度(范围:40%-70%),用于对活啮齿动物进行实验。小鼠在II级安全柜中操作。
    4. 您可以使用持久的爪子纹身在出生后第1天识别出幼崽,皮下注射0.3 mm x 13 mm针头。
  3. 男性气味(来自6只不熟悉的雄性成年OF1小鼠的10克脏污床上用品)
  4. 矿物油(西格玛,法国目录编号:M5904)&nbsp;
  5. Citral(西格玛,法国目录号:W230316)
    注意:柠檬醛有柠檬味。
  6. 70%乙醇溶液
  7. 柠檬醛解决方案(见食谱)

设备

  1. II级安全柜
  2. 定制的声嗅测量仪(如图1所示)
    1. 录音系统由以下部分组成:
      1. 声卡录音机(PreSonus AudioBox iTwo)(图1)。声卡可以由任何其他商业模型替换。调节记录电位计以最大化信噪比并避免过载。
      2. 外差球探测器(其麦克风已使用BNC电缆移入腔室隔离器)(Magenta Bat5 Digital Bat Detector,RSPB,UK)(图1和2)。将外差球探测器设置为鼠标发声的中心频率:在中间调节球探测器的音量控制以避免背景噪音。


      3. 图1.声嗅测量仪。声嗅测量仪是一种在记录小鼠发出的超声波的同时提供异味的系统。它由四个子系统组成:1)录音系统; 2)两个相同的隔离室(仅呈现一个室); 3)气味分配器(嗅觉计); 4)具有自己软件的计算机。



    2. 隔离室(图2):
      1. 排气泵(Schego,目录号:850)
      2. 止回阀(检查)(由Colder Products的两个元件组成;目录号:PLCD220-04和PLCD10004)
      3. 排气流量计(Brooks Instrument,目录号:FR2A13BVBN)


      图2.隔离室隔离室(内部尺寸为18 x 12 x 12 cm)密封,关闭时气密。隔离室由组件制成(8毫米厚的黑色PVC墙用于天花板,地板和侧面,8毫米厚的透明有机玻璃用于门,5毫米直径的橡胶密封用于门,0.1毫米厚的铝涂层有2毫米直径孔的板材,可在DIY商店轻松买到,如Lacrylic商店,法国Bonneuil sur Marne。气味端口和超声波麦克风位于腔室的右侧。气味分配器通过气味端口以受控方式释放气味/空气混合物(图3)。左侧中心的排水孔连接到HEPA排气过滤器(Millex-FG,0.20μm,PTFE疏水物,50mm)。空气通过排气泵,一个止回阀(由Colder Products的两个元件和一个排气流量计(图3)组成)排空,从而避免微生物污染环境。气味剂直接转移到动物设施的排气口。经过所有幼犬的测试,HEPA排气过滤器在实验结束时更换。超声波麦克风在箱内偏移。

    3. 气味分配器(嗅觉计)(图3):
      1. 发射空气泵(Schego,目录号:M2K3)
      2. 空气流量计(Key Instrument,目录号:FR2A14BVBN)
      3. 气味流量计(Brooks Instrument,目录号:FR2A13BVBN)
      4. 气味阀(例如,ASCO,目录号:SCH284A005.12 / DC或Bio-Chem Fluidics,目录号:100P2NC12-05B或等效的常闭电磁夹管阀)
      5. 排气泵(Schego,目录号:850)
      6. C-Flex ®标准管(ID:0.125 OD:0.250)
        注意:管道符合GMP标准。
  3. 注意:用于控制系统的所有电子设备都是定制设备,但很容易被商业化版本的Arduino卡取代: https://www.arduino.cc/en/Main/Products 。


    图3.气味分配器。气味分配器以受控方式分配气味/空气混合物。来自排气泵的空气被传送到空气流量计和气味流量计。该装置允许人们调节气味/空气比(气味为0.3 L / min,空气为2 L / min)。如果气味阀(可以是“ASCO”,“Bio-Chem Fluidics”或等效的常闭电磁阀夹紧阀)关闭,则清洁空气将扩散到腔室中,否则气味将在腔室中均匀地扩散。通过排气泵以1.5L / min的流速将空气抽空到隔离室中。 C-Flex ®标准管(ID:0.125 OD:0.250)用于连接不同的设备。

  4. 个人防护装备(PPE)
    可能包括(但不限于)磨砂,无菌组合,乳胶手套,蓬松帽,通风面罩,防护眼镜和鞋套,具体取决于正在进行工作的动物设施的规定。

软件

  1. 该软件(图4):
    Pups发射超声波发声,频率为40-120 kHz,使用连接到蝙蝠探测器(频率范围10-130 kHz)的超声麦克风检测,将超声波声音转换为可听频率范围。使用探测器的宽带60 kHz输出,使用Audacity开放软件对超声波呼叫进行采样,记录和分析( www.audacityteam.org )。


    图4.录音软件。此图显示了带有录音图像和气味扩散软件的计算机屏幕。通过定制软件确保控制气味的扩散。该软件可以用Node-RED软件和Arduino代替。由于Arduino显示为串行设备,因此可以使用串行输入/输出节点与其进行通信( https: //nodered.org/docs/hardware/arduino )。

  2. GraphPad Prism软件(GraphPad Software,USA)用于数据分析

程序

注意:

  1. 手套应该用于处理老鼠,气味剂,声嗅测量仪及其腔室的所有步骤。
  2. 在将幼崽带入声嗅觉仪之前,准备好气味剂,气室(确保清洁度,电线和气味分配器,蝙蝠探测器和计算机软件的连接性。
  3. 准备一张包含所有动物信息的表格,包括动物编号,足部标记,基因型,治疗和/或接种物,腔室编号,重量, 等 。>
  4. 在测试前五分钟,幼崽从教室移动,最终从殖民地隔离器移到测试室的二级安全柜,在他们的家庭笼子中放置水坝和垃圾。将两只幼崽放入二级安全柜下的声嗅觉仪的两个可拆卸室中(每室一个幼崽);然后将带有幼崽的室放入实验室制造的声嗅觉仪中,放置在BSL-2室内,靠近II级安全柜,用于USV录音。
  5. 声纳米嗅觉仪中的每个测试阶段持续5分钟。在出生后第6天和第8天,小狗可以两两进行测试。
  6. 每天最多只有一个会话,每个会话只包含一种气味。
  7. 每天测试后应将小狗称重(放置在安全柜中的天平),然后再放回巢中。它们应该与母亲分开总共不到30分钟(包括转移到室内,测试,称重和转移到家笼)。

录制USV (图5)


图5.超声波发声的记录和量化。 A和B.超声波呼叫的记录在将幼崽放入声嗅测量仪的测试室后30秒开始。使用连接到球探测器的超声麦克风检测超声波发声,球探测器将超声波声音转换为可听频率范围(从20到20,000Hz)。 C.实验范例。在第一阶段记录超声波排放,没有气味(1分钟),接着是一段气味暴露(1分钟),最后是最后一段废气(1分30秒。这个持续时间允许完全消除含有气味剂的废气)。 D.来自断奶前6日龄幼仔的自发呼叫系列的典型波迹(更多细节,参见Lazarini et al。,2018)。大多数发声(频谱图上持续时间<100ms的发声单位)与呼叫间隔串联产生。 130毫秒


  1. 将含有幼崽的两个室放在声嗅测量计的桌子上。连接声嗅测量仪(SO)和计算机。打开蝙蝠探测器。
  2. 将两个腔室连接到声嗅觉仪后30秒,通过启动录音软件记录USV。同时记录两只幼崽发出的USV,每只幼崽都在自己的密封室内。例程协议如图6所示.USV录制的一个例子是Sound 1(此音频文件描绘了两只2日龄幼崽的USV。)


    图6.在声嗅测量仪中记录USV。实验程序的连续阶段。使用探测器的宽带60 kHz输出,使用Audacity开放软件对超声波呼叫进行采样,记录和分析。

  3. USV录制开始后60秒,启动气味扩散软件。&nbsp;
  4. 气味扩散程序完成后(持续时间:5分钟),在文件名中包含日期,实验编号和所有其他所需信息。
  5. 将幼崽运送到他们的家笼(从室转移到安全柜,称重并转移到家笼的巢中)。&nbsp;
  6. 使用70%乙醇清洁腔室。在间隔5分钟以消除乙醇气味之后,使用与其他幼崽相同的腔室开始下一次测试。&nbsp;
  7. 现在可以使用Audacity软件分析USV记录。分析的一个例子如图7所示。


    图7.嗅觉USV抑制试验的样品数据。 A.实验的时间表。同一窝定时怀孕小鼠中的所有幼鼠在胚胎第13天(E13)在子宫内单独感染,在麻醉下进行胎盘内接种鼠CMV(Smith株)。作为对照(CTL)组,其他定时怀孕小鼠的同一窝中的所有幼仔在E13下在麻醉下在胎盘内注射PBS。在出生后第6天和第8天使用声嗅测量仪分析动物。 B和C.出生后第6天超声波调用柠檬醛气味剂(n = 18 CTL,n = 19 CMV)。 D和E.出生后第8天超声波呼吸男性气味气味(n = 8 CTL,n = 11 CMV)。 P 值通过Wilcoxon匹配对符号秩检验计算。 ** P &lt; 0.01,**** P &lt; 0.0001; B-E中的平均值±SEM。 CTL幼崽分别因接触非社会或社交气味分子(如柠檬醛或雄性气味)而减少呼叫发射。相比之下,先天性CMV感染损害了两种气味引发的超声波呼叫反应,表明病毒引起的嗅觉感知改变(更多细节见Lazarini et al。,2018)。 />

数据分析

使用Audacity开放软件( www.audacityteam.org )手动计算隔离后发出的超声波发声次数。计算每个时间段的超声波发射平均速率(呼叫/分钟):第一个没有任何气味的时间段(1分钟),第二个时期暴露于社交或非社交气味(1分钟)和最后一个时期排气味道(1分钟和30秒)。使用GraphPad Prism分析数据,适当地使用Mann-Whitney或Wilcoxon匹配对符号秩检验。

笔记

  1. 在测试幼仔之前,我们建议通过在它们每个中连续记录2天的相同CTL幼仔来验证两个声 - 嗅觉计室。
  2. 由于该测试是非操作性测试(记录暴露于不同嗅觉线索的幼崽发出的自发USV),因此应使用每组至少8只幼崽来达到分析所需的统计功效。&nbsp;
  3. 当操纵两组动物,一组感染而另一组未感染时,我们建议使用外部标记将两个SO室中的每一个分别用于每组。这应该有助于防止CTL幼崽的任何污染。
  4. Citral加味剂的稀释液应在测试前进行。我们建议在动物设施外的实验室中在通风橱下制备香味,因为防止动物设施中的气味扩散很重要(气味习惯)。
  5. 为了获得更高的重复性,在任何微生物转化气味之前应使用新鲜的男性床上用品。

食谱

  1. 柠檬醛解决方案
    1毫升柠檬醛
    ad 10毫升矿物油稀释液

致谢

所提出的声嗅测量仪的前身(即,基于Slotnick和Bodyak,1999中描述的八通道嗅觉计的小鼠USV抑制任务)由Drs Morgane Lemasson,Gilles共同开发。 Gheusi和Pierre-Marie Lledo(首次发表于Lemasson et al。,2005)。用于开发声嗅测量仪的资金是在巴黎Institut Pasteur(GPF 2015 Microbes&amp; brain“INFECSMELL”)授予FL的资助中提供的。

利益争夺

这些结果是2019年1月18日提交的美国临时专利申请号62/793941的主题,其中FrançoiseLazarini,SébastienWagner和Pierre-Marie Lledo被引用为发明人。

伦理

根据法国法律和符合欧洲共同体理事会指令(2010/63 / UE,法国法律2013-118,2013年2月6日),根据Inserm和巴斯德研究所动物护理的规定,所有动物程序均按照法规进行。委员会。巴斯德研究所的动物实验伦理委员会(CETEA 89)批准了这项研究(2015-0028)。将小鼠圈养在隔离器中,并在法国农业部认可的巴斯德研究所动物设施的II级安全柜中操作,以对活啮齿动物进行实验。

参考

  1. AlAïn,S.,Perry,R。E.,Nunez,B.,Kayser,K.,Hochman,C.,Brehman,E.,LaComb,M.,Wilson,D。A. and Sullivan,R。M.(2017)。 对发育中的幼鼠的母体气味进行神经行为评估:对社交缓冲的影响。 Soc Neurosci 12(1):32-49。
  2. Bell,R。W.,Smotherman,W。P.(1980)。 母亲影响和早期行为。 Spectrum Publ。 Inc.,London,pp.105-133。
  3. Bodyak,N。和Slotnick,B。(1999)。 自动嗅觉仪中小鼠的表现:气味检测,鉴别和气味记忆。 Chem Senses 24(6):637-645。
  4. Branchi,I.,Santucci,D.,Vitale,A。和Alleva,E。(1998)。 婴儿实验室老鼠的超声波发声:不同条件下的初步光谱表征。 Dev Psychobiol 33(3):249-256。
  5. Brouette-Lahlou,I.,Vernet-Maury,E。和Vigouroux,M。(1992)。 幼犬超声波呼叫在Wistar大鼠特定母体行为中的作用:幼仔的肛门生殖器舔。 / a> Behav Brain Res 50(1-2):147-154。
  6. Brunelli,S.A.,Shair,H.N。和Hofer,M。A.(1994)。 大鼠幼仔的低温发声( Rattus norvegicus )引出并指导母体搜索行为。 J Comp Psychol 108(3):298-303。
  7. Brunjes,P。C.和Alberts,J.R。(1979)。 嗅觉刺激诱导蜷缩在大鼠幼崽中的偏好。 J Comp Physiol心理学 93(3):548-555。
  8. Castellucci,G.A.,Calbick,D。和McCormick,D。(2018)。 鼠标超声波发声的时间组织。 PLoS One 13(10):e0199929。
  9. Hofer,M。A.和Shair,H。N.(1991)。 三叉神经和嗅觉通路介导大鼠幼仔的隔离窘迫和伴随舒适反应。 Behav Neurosci 105(5):699-706。
  10. Hofer,M.A.,Shair,H.N。和Brunelli,S.A。(2002)。 大鼠和小鼠幼仔的超声波发声。 Curr Protoc Neurosci >第8章:单元8.14。
  11. Hongo,T.,Hakuba,A.,Shiota,K。和Naruse,I。(2000)。 由遗传性脑瘫小鼠嗅觉系统缺陷引起的哺乳功能障碍。 Biol Neonate 78(4):293-299。
  12. Kobayakawa,K.,Kobayakawa,R.,Matsumoto,H.,Oka,Y.,Imai,T.,Ikawa,M.,Okabe,M.,Ikeda,T.,Itohara,S.,Kikusui,T., Mori,K。和Sakano,H。(2007)。 小鼠嗅球中的先天与学习气味处理。 自然 450(7169):503-508。
  13. Lazarini,F.,Katsimpardi,L.,Levivien,S.,Wagner,S.,Gressens,P.,Teissier,N。和Lledo,P.M。(2018)。 先天性巨细胞病毒感染可改变小鼠听力恶化前的嗅觉。 J Neurosci 38(49):10424-10437。
  14. Lemasson,M.,Delbé,C.,Gheusi,G.,Vincent,J。D. and Lledo,P.M。(2005)。 使用超声波发声评估用3-甲基吲哚治疗的小鼠幼崽的嗅觉检测。 Behav Processes 68:13-23。
  15. Noirot,E。(1974)。 通过未接种的幼犬接触超声波的原始雌性小鼠筑巢。 Anim Behav 22:410-420。
  16. Perry,R.E.,Al Ain,S.,Raineki,C.,Sullivan,R。M. and Wilson,D.A。(2016)。 气味享乐的发展:支持经验依赖的回路发育过程,支持大鼠的母体和捕食者气味反应。< / a> J Neurosci 36(25):6634-6650。
  17. Sarnat,H。B.和Yu,W。(2016)。 人类嗅球的成熟和发育不全。 Brain Pathol > 26(3):301-318。
  18. Shair,H。N.,Masmela,J.R。和Hofer,M.A。(1999)。 嗅觉对增强和抑制大鼠超声发声的影响。 Physiol Behav 65(4-5):769-772。
  19. Slotnick,B。和Restrepo,D。(2005)。 用鼠标进行嗅觉测定。 Curr Protoc Neurosci 第8章: 8.20单元。&nbsp;
  20. Smith,J。C.,and Sales,G.D。(1980)。啮齿类动物的超声波行为和母婴相互作用。在:母亲影响和早期行为。贝尔,R。W.和Smotherman,W。P.(编辑)。 SP医学和科学书籍,纽约,第105-133页。
  21. Stickrod,G.,Kimble,D。P.和Smotherman,W。P.(1982)。 在子宫内味觉/气味厌恶调节大鼠。 Physiol Behav 28(1):5-7。
  22. Szentgyörgyi,H.,Kapusta,J。和Marchlewska-Koj,A。(2008)。 银行田鼠的超声波呼叫被隔离并暴露于寒冷或窝臭。 Physiol Behav 93(1-2):296-303。
  23. Teicher,M。H.和Blass,E。M.(1976)。 哺乳新生大鼠:通过乳头灌洗消除,通过小狗唾液恢复。 Science 193(4251):422-425。
  24. Yang,M。和Crawley,J。N.(2009)。 对小鼠嗅觉的简单行为评估。 Curr Protoc Neurosci 第8章:单元8.24。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wagner, S., Lledo, P. and Lazarini, F. (2019). Assessing Olfaction Using Ultrasonic Vocalization Recordings in Mouse Pups with a Sono-olfactometer. Bio-protocol 9(4): e3170. DOI: 10.21769/BioProtoc.3170.
  2. Lazarini, F., Katsimpardi, L., Levivien, S., Wagner, S., Gressens, P., Teissier, N. and Lledo, P. M. (2018). Congenital cytomegalovirus infection alters olfaction prior to hearing deterioration in mice. J Neurosci 38(49): 10424-10437.
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