In this study, we used 26 anophthalmic mice (10 females and 16 males) and 20 sighted mice (four females and 16 males) in total. All mice were bred and housed in mixed cages of 3–6 blind and sighted individuals in the animal facility. Standard and constant housing conditions were applied including a 14/10 h light/dark cycle, a free and ad libitum access for water/food, and a controlled room temperature set at 21°C and 40–60% humidity. Pups were tattooed on the paw for identification at PND 1. In the ZRDBA strain, breeding a blind mouse with a sighted mouse generates litters comprising half the pups born blind (homozygous, results in the absence of eyes and optic nerves), and the other half born sighted (heterozygous). All experimental procedures were approved by the animal care committee of Université du Québec à Trois-Rivières in accordance with the Canadian Council on Animal Care guidelines.

Behavioral experiments carried out during the light phase between 9:00 a.m. and 5:00 p.m. All mice were assayed to the following behavioral tests conducted at three developmental stages (see Figures 1, 2), namely, (1) at PND 7, the maternal isolation-induced pup USV test, followed by the home odor discrimination and preference test; (2) at PND 30–35, the juvenile social test; and (3) at 2–3 months, the female urine-induced USVs and scent-marking behavior test. At PND 7, all the pups from all litters were assayed to the maternal isolation-induced pup USVs test and the home odor discrimination and preference test (n = 26 blind and 20 sighted mice). At PND 35, all of the mice from all litters were tested as experimental mice in the juvenile social test, except for mice used as social stimuli (n = 15 blind and 14 sighted). All males were assessed in female urine-induced USVs and scent-marking behavior (n = 16 blind and 16 sighted mice).

All behavioral recordings and codings were performed using Ethovision XT software (Noldus, VA, USA). To record USVs' rates, an ultrasonic microphone was suspended above or attached to the wall of the experimental cage according to the behavioral tests. USVs' recordings were processed and analyzed through the UltraVox XT system (Noldus Information Technology, The Netherlands). The system can record the full spectrum of sound and has a maximum frequency of 160 kHz.

We used SPSS 22.0 (IBM, Armonk, NY, USA) for statistical analysis. We verified normal data distribution using the Shapiro-Wilk/Kolmogorov-Smirnov test. For data not normally distributed, we used the Mann-Whitney test to compare the experimental groups. All data are shown as mean ± SE. Regarding the maternal isolation-induced pup USV test, we computed a repeated-measures ANOVA with time (two levels—isolation 1 and isolation 2) as within-subject factor and group (two levels—anophthalmic mice and sighted mice) as between-subject factor. Regarding the home odor discrimination and preference, we computed odor preference of each mouse as a difference index: time spent above home bedding odor—clean bedding odor (Meyer and Alberts, 2016). Consequently, we compared odor preference score and USV rates between the groups with Mann-Whitney U-tests for independent samples. Regarding the juvenile social and the scent-marking behavior tests, we compared both groups using a one-way MANOVA (independent groups—two visual groups; dependent variables—measures within a test). We examined correlations between variables for each group using Spearman's correlation. For all analyses, we set the significance level at p < 0.05, with appropriate corrections for multiple comparisons (Bonferroni's correction).

Repeated-measures ANOVA analysis revealed a significant effect of time [F_(1,36) = 6.885, p = 0.013], but we did not find any effect of visual status [F_(1,36) = 0.854, p = 0.362] or the interaction time ^* visual status [F_(1,36) = 2.973; p = 0.094] (Figure 3).

Given that data for odor preference index and USV rates did not pass the Shapiro-Wilk normality test (W = 0.719, p < 0.001 and W = 0.852, p = 0.015, Figure 4), we performed Mann-Whitney tests. There was no significant effect of odor preference index and USVs' rates between both groups (U = 155, p = 0.888; U = 197.5, p = 0.236). In addition, no correlation was found between the number of USVs and the odor preference index in both phenotypes [blind: r(16) = −0.147, sighted: −0.383, p > 0.05].

One-way MANOVA test revealed a significant effect of visual status on behavioral variables measured [F_(9,19) = 2.925, p = 0.023, Figure 5]. Specifically, blind mice exhibited less time spent in sniffing [F_(1,27) = 15.635, p < 0.001] and in following the congener [F_(1,27) = 6.499, p = 0.017] compared to sighted mice. Moreover, blind mice spent more time exploring the cage alone [F_(1,27) = 3.977, p = 0.046]. Overall, Student's t-tests confirmed that blind mice engaged less social behaviors [t_(1,27) = 4.772, p < 0.001] and consequently more frequent non-social behaviors [t_(1,27) = −5.419, p < 0.001]. Nevertheless, the total number of USVs emitted was similar between both groups [F_(1,27) = 0.202, p = 0.657].

In addition, we did not find any correlation between the total number of USVs and any social or non-social behaviors in both phenotypes [blind: r(16) = −0.335 to 0.253, sighted: −0.473 to 0.399, p > 0.05]. In blind mice, time spent exploring alone the environment was positively correlated with distance traveled [r(16) = 0.526, p = 0.044] and negatively associated with time spent sniffing the congener [r(16) = −0.601, p = 0.018], following the congener [r(16) = −0.532, p = 0.041], and sitting alone [r(16) = −0.561, p = 0.030]. In sighted mice, time spent following the congener was positively correlated with time spent sniffing this latter [r(16) = 0.572, p = 0.033] and negatively correlated with time spent sitting close to it [r(16) = −0.554, p = 0.040]. Besides, time spent exploring the cage in sighted mice was negatively associated with time spent self-grooming [r(16) = −0.688, p = 0.007].

The one-way MANOVA revealed a significant effect of visual status on behavioral variables measured [F_(6,25) = 2.560, p = 0.040, Figure 6]. Specifically, blind mice were faster to sniff the urine spot and spent more time sniffing it [F_(1,30) = 8.934, p = 0.006, F_(1,30) = 4.580, p = 0.040, respectively]. Additionally, blind mice emitted USV faster than their sighted counterparts [F_(1,30) = 4.679, p = 0.039]. No difference was demonstrated in both groups in terms of total numbers of USVs, distance traveled, and total surface marked [F_(1,30) = 0.715; F_(1,30) = 0.008; F_(1,30) = 0.404, p > 0.05, respectively].

In addition, no correlation was found between the total number of USVs and any behavioral variables in blind mice [r(16) = −0.462 to 0.370, p > 0.05], whereas a positive correlation was found between the total number of USVs and (1) the total numbers of scent marks and (2) the latency before emitting USVs' calls in sighted mice [r(16) = 0.593, p = 0.015; r(16) = 0.535, p = 0.033, respectively]. Finally, the more sighted mice sniff the urine spot, the more they mark it [r(16) = 0.620, p = 0.010].

In blind mice, there was no correlation between the total number of USVs calls produced in the pup isolation, odor preference, social juvenile, and scent-marking tests. In sighted mice, a positive correlation was found between the total number of USVs emitted by PND 7 pups during the first 5-min isolation and during the scent-marking test in male adults [r(16) = 0.599, p = 0.014, Figure 7].

Isolation-induced USV has been widely employed as a marker for distress/anxiety in pups' rodents. USVs emitted by pup mice and rats are a spontaneous response to isolation from their mother (Barnes et al., 2017). Our results revealed no differences in the number of USVs emitted during the first and/or the second maternal isolation between sighted and blind pups. At PND 7, visual impairment had no incidence on isolation induced-vocalization behavior. Both groups having their eyes closed at this early developmental stage [the eyes opening in sighted mice occurs around PND 11–13 (personal observations)], we can assume that the visual status of both groups is similar. This indicates that the possible neuroanatomical differences between sighted and anophthalmic pups, such as a lack of optic nerve (Touj et al., 2019), have no impact on USVs' production behavior at PND 7. In this study, we also observed no difference between anophthalmic and sighted pups' calls emission during a second maternal isolation phase compared to the first phase, both groups emitting less calls in the second period. Hence, we did not observe the “maternal potentiation” phenomenon, namely, a higher acoustic response induced by a second isolation phase, which has been described previously in several studies in rats (Hofer et al., 1994; Shair, 2007) and in Swiss-Webster mice at PND 7–8 (Scattoni et al., 2008, 2009). Our results, however, are consistent with other studies, which showed no or decreasing number of USVs produced during the reisolation period in C57BL/6J mice (Barnes et al., 2017) and guinea pigs (Hennessy et al., 2006), respectively.

Besides USVs' emission, we also observed that both anophthalmic and sighted 7-day-old pups had similar olfactory behaviors. Indeed, both groups were able to discriminate and preferred exploring their home bedding's compared to the clean bedding's odors. These results are consistent with several studies showing that 9- to 13-day-old mice express their preference for their own nest to a clean one by searching and reaching the home nest/bedding area and spending more time on it (Scattoni et al., 2008; Lo Iacono et al., 2021; Premoli et al., 2021). It has also been suggested that, prior to the opening of the eyes pups' exploratory behavior is exclusively driven by olfactory cues (Freeman and Rosenblatt, 1978). Our findings indicate that both genotypes displayed the same odor performance in terms of odor discrimination and preference.

Taken together, our congenital blindness mouse model did not present alteration of neither the number of isolation-induced USVs emitted nor social olfactory behaviors at PND 7.

During social play, the pairs of blind-sighted and sighted-sighted juvenile mice displayed a similar number of USVs. Interestingly, social behavior was altered in the blind mice, with a heightened time exploring the cage to the detriment of sniffing and following the congener. No differences between sighted and anophthalmic juvenile mice were observed otherwise in self-grooming, social sitting, and sitting-alone behaviors. Consistent with previous studies (Klein and Brown, 1969; Dyer and Weldon, 1975; Iura and Udo, 2014), we recently reported that congenitally blind mice exhibited an enhanced motivational level to explore a new environment, such as seen in the open-field, Elevated Plus Maze (EPM), and Forced Swim tests, compared to sighted controls (Bouguiyoud et al., 2022). The hyperactivity observed in blind mice when exploring a novel environment may reflect a compensatory mechanism by which the lack of visual information is counterbalanced by gathering and memorizing information from the physical world before it becomes familiar (Iura and Udo, 2014). Accordingly, blind animals may require a longer habituation period to explore the new environment. This could explain their increased general exploratory behavior despite the presence of a social stimulus mouse.

Furthermore, this apparent preference for exploring the environment over engaging in social interactions with a congener resembles the social deficits described in mouse models of autism (Moy et al., 2004; McFarlane et al., 2008; Silverman et al., 2010). Animal models of autism showed social behavior deficits such as less time sniffing, grooming, and following an unfamiliar congener (Brodkin, 2007; McFarlane et al., 2008) and increased time allocated to self-grooming and inactivity (McFarlane et al., 2008).

Interestingly, in humans, a link between congenital blindness and autism was proposed based on the dramatic increased prevalence autism in individuals suffering visual impairments (Jure et al., 1991; Hobson et al., 1999; Hobson, 2011; Suhumaran et al., 2020). Moreover, children with profound visual impairment show delays in the development of joint attention behaviors, such as sharing or talking about their interests to others (Tadić et al., 2010; Dale et al., 2014). However, social behavioral differences in visually impaired individuals may be caused by specific sensory limitations, as suggested by Chokron et al. (2020), and the link between early visual deprivation, social communication, and social behavior throughout development is still unclear in humans and non-human animal models.

Taken together, the similar amount of calls in the pairs of juveniles with a lower amount of social interaction in the blind-sighted mice indicates that either (1) blind-sighted pairs of mice produced higher amounts of USVs in a shorter total duration of social interactions than the sighted-sighted ones or (2) the calls were emitted during social and non-social exploratory behaviors. It should be noted that blind animals developed ultrasonic echolocation abilities to help them explore efficiently their surroundings (Griffin, 1944; Dufour et al., 2005; Schenkman and Nilsson, 2010; Thaler et al., 2011; Kupers and Ptito, 2014). Future studies should track each animal and record their acoustic behavior emitted specifically during social behaviors, such as allogrooming, sniffing, following, or specific non-social behaviors, such as exploration.

Adult male mice exposed to females' urinary marks show a fast approach and prolonged sniffing behavior, emit ultrasonic vocalizations, a phenomenon known as the “ultrasonic courtship” vocalizations (Nyby, 1983; Holy and Guo, 2005; Arakawa et al., 2009; Wöhr and Schwarting, 2013), and deposit a large number of scent marks (Hurst, 1989; Arakawa et al., 2007), especially around the urinary source (Roullet et al., 2011). In this study, congenitally blind mice exhibited shorter latency to first sniff and higher duration sniffing the female urine spot, together with a shorter latency to emit the first female urine-induced USV call. The total number of calls, however, was similar between blind and sighted mice. Previous studies using the buried food test showed that blind rodents had a shorter latency to detect environmental odorants such as an appetent olfactory source than sighted congeners in congenitally blind ZRDBA mice (Touj et al., 2021) in visually deprived C57BL6 mice and rats (Zhou et al., 2017). To the best of our knowledge, our results highlight for the first time the importance of social olfactory cues (female urine) detection and their impact on acoustic behavior in anophthalmic male mice. In addition, histological and structural imaging studies reported larger olfactory bulbs and hypertrophy of brain areas involved in the olfactory processing (e.g., the anterior olfactory nucleus, the olfactory tubercle, the piriform cortex) in anophthalmic ZRDBA mice, supporting their heightened olfactory performance (Touj et al., 2020, 2021).

Accordingly, a longer time spent exploring the urinary source observed in blind mice may indicate a high attentional/motivational process toward odor stimuli. Similarly, increased attentional processes were found in our congenitally blinded mice in a two-odor choice test in response to positive odors (i.e., peanut butter and vanilla scent) compared to sighted mice (Touj et al., 2020). Thereby, visually impaired humans and non-human animals pay more attention to non-visual cues, processing them more efficiently compared to sighted individuals, which might enhance non-visual abilities (Kujala et al., 1997; Liotti et al., 1998; Hugdahl et al., 2004; Collignon et al., 2006; Collignon and De Volder, 2009; Ferdenzi et al., 2010; Beaulieu-Lefebvre et al., 2011; Pigeon and Marin-Lamellet, 2015; Topalidis et al., 2020).

Regarding the scent-marking behavior, the total surface marked was not different between sighted and blind adult mice in the whole cage and at 10 cm² around the female urine drop. Dominant male mice tend to mark more than the subordinate ones (Desjardins et al., 1973; Rich and Hurst, 1998), so this result suggests that congenital blindness does not affect social ranking and reproductive-like behavior (both phenotypes are housed within the same cage). Further studies should examine in detail male social hierarchy in the context of sensory deficits such as visual deprivation. Finally, we reported a similar locomotor activity between the two groups in this test, which was also observed in Touj et al. (2020) when mice are assayed to an olfactory test.

Interestingly, in sighted, but not in the blind, adult male mice, there was a significant correlation between the number of female urine-induced calls and the total number of scent marks produced. This discrepancy may suggest an altered communication behavior in blind male mice in response to female urine, with a weaker coherence between the communication modalities (call emission and scent marking). This result is reminiscent of previous studies in a mouse model for autism, where the correlation between scent marking and USV emission appeared incoherent in the autism-like male group compared to the controls (Wöhr et al., 2011).

In conclusion, blind and sighted adult male mice exhibited differences in social communication and behavior in response to female urine stimuli.

The longitudinal analysis of the acoustic and related social behaviors across the three developmental stages examined in this study, namely, early, juvenile, and adult stages, show that in sighted mice, but not in the blind, the number of USVs emitted during the first maternal isolation is correlated with the total number of scent marks in the adult. The calls emitted by the mice at PND 7 during the isolation might indicate a high level of arousal, attention, and motivation in the pups, which may then be reflected in and predict the scent-marking behavior in the adult.

It should be noted that both behaviors are crucial for the social life in the mouse, and this correlation may indicate a common biological foundation and neurological pathway (Demir et al., 2020). In contrast, social play behavior in the juvenile has been described as a unique category of behavior and does not predict future adult social, sexual, or aggressive behavior (Vanderschuren et al., 1997).