Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism

Behavioural Brain Research 270 (2014) 159–164

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Short Communication

Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism Anes Ju a,b , Kurt Hammerschmidt c , Martesa Tantra a,b , Dilja Krueger d , Nils Brose b,d , Hannelore Ehrenreich a,b,∗ a

Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany DFG Center for Nanoscale Microscopy & Molecular Physiology of the Brain (CNMPB), Göttingen, Germany c Cognitive Ethology Laboratory, German Primate Center, Göttingen, Germany d Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany b

h i g h l i g h t s • • • •

Nlgn4−/− mice are a construct valid autism model, now confirmed in pups and juveniles. USV in pups on brief maternal separation revealed remarkable gender and mild genotype effects. USV in juveniles on exposure to an anesthetized female was reduced in Nlgn4−/− mice. Neonatal development from PND4 to 21 did not yield further differences between genotypes.

a r t i c l e

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Article history: Received 26 March 2014 Received in revised form 6 May 2014 Accepted 12 May 2014 Available online 20 May 2014 Keywords: Neuroligin-4 C57BL/6J Ultrasound or ultrasonic vocalization Neonatal milestones Neonatal development Gender

a b s t r a c t Neuroligin-4 (Nlgn4) is a member of the neuroligin family of postsynaptic cell adhesion molecules. Lossof-function mutations of NLGN4 are among the most frequent, known genetic causes of heritable autism. Adult Nlgn4 null mutant (Nlgn4−/− ) mice are a construct valid model of human autism, with both genders displaying a remarkable autistic phenotype, including deficits in social interaction and communication as well as restricted and repetitive behaviors. In contrast to adults, autism-related abnormalities in neonatal and juvenile Nlgn4−/− mice have not been reported yet. The present study has been designed to systematically investigate in male and female Nlgn4−/− pups versus wildtype littermates (WT, Nlgn4+/+ ) developmental milestones and stimulus-induced ultrasound vocalization (USV). Neonatal development, followed daily from postnatal days (PND) 4 to 21, including physical development, neurological reflexes and neuromotor coordination, did not yield any differences between Nlgn4−/− and their WT littermates. USV in pups (PND8–9) in response to brief separation from their mothers revealed remarkable gender effects, and a genotype influence in females regarding latency to first call. In juveniles (PND22–23), USV monitoring upon exposure to an anesthetized female intruder mouse uncovered a clear genotype effect with reduced USV in Nlgn4−/− mice, and again a more prominent phenotype in females. Together, these data support an early manifestation of communication deficits in Nlgn4−/− mice that appear more pronounced in immature females with their overall stronger USV as compared to males. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The discovery of heritable forms of autism spectrum disorder (ASD) involving mutations in genes encoding neuroligin-4X (NLGN4X), neurexin-1 (NRXN1), neuroligin-3 (NLGN3), SHANK2,

∗ Corresponding author at: Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany. Tel.: +49 551 3899615; fax: +49 551 3899670. E-mail address: [email protected] (H. Ehrenreich). http://dx.doi.org/10.1016/j.bbr.2014.05.019 0166-4328/© 2014 Elsevier B.V. All rights reserved.

and SHANK3, has revealed autism as a disease of the synapse and led to the development of urgently needed mouse models with proven construct validity [1–7]. It has been estimated very conservatively (probably underestimated) that these so-called ‘monogenic forms’ of ASD are relevant in 1–2% of the patients [8]. We previously reported a remarkable autism-like phenotype in Nlgn4−/− mice of both genders, comprising deficiencies in ultrasound communication, social interaction and competence as well as increased stereotypies and repetitive behaviors [1,9]. Neuroligin-4 (Nlgn4) is a postsynaptic cell adhesion molecule that forms trans-synaptic complexes with presynaptic neurexins and regulates maturation,

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plasticity and function of synapses. It is abundantly expressed in hippocampus, cortex and septum and lowest in brainstem and cerebellum. During murine brain development, Nlgn4 expression increases from low levels at embryonic and early postnatal phases to a plateau at 3 weeks after birth when synapse formation has reached maturity [1]. Since autism is usually diagnosed in children before the age of 3 years, evaluation of neurodevelopmental milestones including early communication skills in neonatal mice up to the age of 3–4 weeks is crucial for further characterization of mouse models of autism (for review see [10]). One previous study investigating Nlgn4−/− pups and juvenile mice was based on pooled genders and failed to find any autism-relevant phenotype [11]. Therefore, the present study has been designed to more extensively assess larger numbers of Nlgn4−/− mice and their WT littermates, separately for both genders regarding (1) neurodevelopmental milestones including physical growth, neurological reflexes and neuromotor coordination from postnatal day (PND) 4 up to PND21, and (2) ultrasound vocalization (USV) in response to defined stimuli not only in pups (PND8/9) but also in juveniles (PND22–23). We report here in mice of both genotypes normal achievement of neurodevelopmental milestones as well as gender differences in pup and juvenile USV. Importantly, as autism-relevant phenotype in Nlgn4−/− mice, an USV deficiency in pups and even stronger in juveniles was found. 2. Methods 2.1. Mice All experiments were approved by the local animal care and use committee. Male and female Nlgn4 null mutant mice (Nlgn4−/− ) and wildtype littermates (WT) with a C57BL/6J genetic background were used [1,9]. They were derived from revitalized frozen embryos that originated from a C57BL/6J-SV129 × Nlgn4−/− mutant line that had been backcrossed into C57BL/6J for 6 generations. After revitalization, Nlgn4−/+ mice were bred with C57BL/6J for 2 further generations, and then Nlgn4−/+ mice were interbred for colony expansion to generate the mice for the present experiments. Approximately 2 weeks after pairing, females were individually housed and carefully inspected twice daily for pregnancy and delivery. The day a litter was first observed was scored as day 0 for that litter. After birth, animals were kept untouched in the home cage with their mothers until PND3 when animals were tagged in their feet using non-toxic animal tattoo ink. The ink was inserted subcutaneously through a 30 gauge hypodermic needle tip into the center of the paw. Experiments were performed by a trained observer, unaware of the genotype (‘blinded’). Litters used for experiments contained 6–10 pups, with 19 litters employed for tests of neurodevelopmental milestones and 15 litters for pup and juvenile vocalization studies. The battery of tests performed provides an assessment of physical and neurodevelopmental milestones as well as neuromotor coordination throughout the neonatal period. The parameters measured are expressed and maturing at different periods throughout the first 21 days of life [12–15]. 2.2. Neurodevelopmental milestones Neonatal assessments comprise (i) maturation readouts describing physical development, (ii) neurodevelopmental measures based on neurological reflexes, and (iii) the achievement of neuromotor coordination. 2.2.1. Maturation readouts Body weight development and the opening of eyes and ears are monitored daily.

2.2.2. Neurodevelopmental measures 2.2.2.1. Placing response. Pups are suspended in the air by grasping the pup gently around the trunk, making sure that none of the paws touched a solid surface. A thin metal bar is put in contact with the back of a paw. Starting from PND4, it is monitored with one trial per day whether the paws are raised (proper response). Criterion is reached when pups show the proper response on 2 consecutive days. 2.2.2.2. Surface righting reflex. Animals are placed on their back on a surface and then released. The time needed for each pup to right itself is recorded and the performance is monitored twice daily, starting from PND4. Criterion is reached when the pup can right itself within 2 s in both trials on 2 consecutive days. 2.2.2.3. Cliff avoidance. The pups are observed daily with one trial from PND6 until pup shows retraction (cliff avoidance reflex) within 10 s after being placed on an edge, with forepaws and nose just over the edge. Criterion is reached when pups show cliff avoidance reflex within 10 s on 2 consecutive days. 2.2.2.4. Negative geotaxis reflex. The pups are observed daily with one trial from PND7 by placing them on an inclined plane (30◦ angle) with head facing downwards. The time needed for the pup to change its orientation, so that its head faced up the incline (proper response) is measured. Response of each pup is observed for 30 s. Criterion is reached when the proper response appears before 30 s on 2 consecutive days. 2.2.2.5. Ear twitch. The cotton tip of an applicator is pulled out and the tip twisted to form a fine filament. The filament is gently brushed against the tip of the ear for 3 times. Criterion is reached when pups show the proper response of flattening the ear against the side of the head on 3 consecutive days. 2.2.2.6. Tactile startle. A puff of air (e.g. experimenter’s breath) is gently applied to the pups, starting on PND10. Criterion is reached when the proper response (jumping or running) is observed on 3 consecutive days. 2.2.2.7. Air righting reflex. The pup is held upside down with 2 fingers holding either side of the head and 2 fingers holding the hind quarters approximately 10 cm over a cage containing 5 cm of shavings. The pup is released and its position upon landing observed for one trial, starting from PND10 until criterion is reached, when the pup is able to turn around and land on the 4 paws over 3 consecutive days. 2.2.3. Neuromotor coordination measures 2.2.3.1. Open field traversal. The pup is placed in the center of a 13 cm circle and the time needed to escape off the circle is recorded, starting from PND10. The trial is terminated when after 30 s the pup remained in the circle. Criterion is reached when the time to move off the circle is <30 s on 2 consecutive days. 2.2.3.2. Wire suspension. Pups are forced to grasp a 3 mm wire and hang from it on their forepaws. Testing starts on PND10 onward until pups are able to hold the wire for 30 s. Criterion is reached when the pup is able to hang for 30 s on 2 consecutive days. 2.3. Ultrasound vocalizations in neonatal and juvenile mice Ultrasonic vocalizations (USV) of pups were recorded on PND8/9 to measure their reaction to brief isolation (3 min) from their mothers (isolation test). Only few animals were tested on PND8. Exploratory separate analysis of mice tested on PND8/9 versus

A. Ju et al. / Behavioural Brain Research 270 (2014) 159–164

mice tested on PND9 only did not yield any appreciable differences in USV results (data not shown), in agreement with previous literature [16]. For juvenile mice, recording was performed on PND22–23 measuring the response upon exposure to an anaesthetized female intruder [9]. For neonatal recording, pups were selected randomly from their litter, weighed and placed directly from the nest in the home cage into a soundproofed custom-made plastic box (diameter 13.5 cm) that contained for each pup a fresh paper towel that covered the floor of the box. This plastic box was located in a room with a constant ambient temperature of 22 ◦ C. An ultrasound microphone (UltraSoundGate CM16) fixed in the lid of the box 12 cm above the bottom was connected to a preamplifier (UltraSoundGate116, Avisoft Bioacoustics, Berlin, Germany), coupled to a notebook computer. Juvenile USV were recorded from the home cage of the resident mouse (male or female). After 1 min of habituation of the test mouse within its home cage in the test room, the mouse was exposed to an anesthetized female intruder mouse and USV were recorded for 3 min. The sampling frequency of 300 kHz resulted in a frequency range of 150 kHz. Because background noise below the frequency range of the produced USVs can have a negative influence on the estimations, we applied a high pass FIR filter of 35 kHz. USV were separated from other sounds using the whistles detection algorithm of Avisoft-SASLab 5.2 (Avisoft Bioacoustics, Berlin, Germany) with following selection criteria: Possible changes per step = 4 (4687 Hz), minimal continuity = 5 ms (neonates) and 8 ms (juveniles), possible frequency range = 35–150 kHz. These criteria were positively evaluated in former studies of pup and adult mouse vocalizations [1,17]. In addition, all estimations of the whistle detection algorithm were visually controlled by an experienced observer, because in rare cases the program can select other sounds such as toe clicking, sniffing or high frequency background erroneously as USVs. Total number, total duration of calls, latency to vocalize (cut-off 30 s) and duration of a single call per recording session as well as general frequency parameters of USV were measured. Emission of ≤10 calls was taken as pre-defined exclusion criterion for determining duration of calls and mean peak frequency. Start, end, maximum and mean of the peak frequency (PF) and the location of maximum PF were estimated. As typical whistles concentrate their sound energy nearly into one single amplitude peak, peak frequency corresponds to the fundamental frequency, although it is difficult to prove as long as no harmonics can be detected. Overall, controlling for systematic changes of USV results over time by plotting quality control charts, like ImR or cumsum charts is routinely performed. 2.4. Statistical analysis Data are presented as mean ± S.E.M. For USV analysis, we excluded statistical outliers (as determined by Grubb’s test), mice with missing data, complete absence of calls or emission of ≤10 calls or values >30 s for latency to first call (in total, exclusions were rare and equally distributed over genotypes). ANOVA was used to check the overall effect of genotype and gender separately for the respective time point of USV experiments. Two-tailed student’s ttests for independent samples were employed for post hoc testing. A p value of <0.05 was considered statistically significant. 3. Results 3.1. Nlgn4−/− mice of both genders accomplish body weight gain and neurodevelopmental milestones comparable to their WT littermates There was no genotype difference in either gender regarding body weight curves obtained from daily measurements starting on

161

PND4 until PND21 (Fig. 1A and B). Similarly, the days to reach criterion for each developmental milestone as well as the respective developmental time curves for all examined readouts were equal among genotypes for both genders (Fig. 1C and D and Table 1). 3.2. Ultrasound vocalization (USV) monitoring reveals genotype effects and gender differences As USV readouts, number and total duration of calls, mean call duration and latency to first call, as well as frequency parameters were registered. In pups, a considerable difference between genders became obvious, with females showing mainly a longer duration of calls (Fig. 2, left column). At this time point, a genotype effect in females was noted only with respect to latency to first call. In contrast, juvenile mice displayed a significant genotype difference for most USV items, again more prominent in females, where also frequency parameters differed between genotypes (Table 2 and Fig. 2, right column). At the juvenile stage, however, the gender difference appeared less pronounced as compared to that observed in pups. This is in agreement with a transient early gender effect that later turns into the comparable USV of both genders found in adulthood [9]. Overall, even though the USV-inducing stimuli are different at the two time points, there appears to be an effect of age on number of calls in WT (p = 0.022; repeated measures ANOVA), with juveniles having more pronounced USV (male: n = 17, 208.71 ± 46.81; female: n = 17, 290.41 ± 39.20) as compared to pups (male: n = 19, 139.11 ± 18.14; female: n = 17, 181.06 ± 27.70). This difference was completely absent in Nlgn4−/− mice (p = 0.486), with the number of calls being similar between juveniles (male: n = 16, 135.00 ± 29.04; female: n = 13, 160.92 ± 35.70) and pups (male: n = 15, 121.93 ± 21.52; female: n = 13, 135.07 ± 36.04). Despite the different USV-provoking stimuli, these results may still point to a delayed or lacking maturation of communication skills in Nlgn4−/− mice. 4. Discussion The present paper systematically investigated neurodevelopmental milestones in male and female pups and juveniles of a construct-valid mouse model of human ASD, Nlgn4−/− mice. Whereas body weight gain, physical development, maturation of reflexes and neuromotor coordination in both genders from PND4 through 21 were indistinguishable between genotypes, there was a remarkable deficiency in juvenile communication as evaluated by stimulus-induced USV. This communication defect was more prominent in females and started already with an increased latency to the first call in female pups. Interestingly, at the pup level, gender differences independent of the genotype are most obvious, with females demonstrating stronger USV. This gender difference lightens in juveniles and disappears in adults [9]. Interestingly, an earlier maturation of verbal skills is also known for female as compared to male babies [18]. These facts together with our observation in Nlgn4−/− mice clearly emphasizes the necessity to investigate genders separately even at this early age. The absence of neurodevelopmental abnormalities in the present cohort up to PND21 is in agreement with an earlier report where smaller numbers of mice, with genders pooled, were followed up to PND14 [11]. Also the lack of significant effects of genotype on USV until PND12 described by these authors could here be widely reproduced. In contrast, these authors did not see gender differences in pup USV [11]. Moreover, they did not check for genotype effects in USV at the juvenile stage where we obtained a highly significant reduction in Nlgn4−/− mice.

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Fig. 1. Assessment of neurodevelopmental milestones in Nlgn4−/− mice and their WT littermates from PND4 through 21. The upper panels display the results for male (Nlgn4−/− n = 25; Nlgn4+/+ n = 19), the lower row for female mice (Nlgn4−/− n = 15; Nlgn4+/+ n = 19). (A and B) Body weight curves; (C and D) Days to reach the milestone criterion in readouts of physical development, reflex maturation and neuromotor coordination. Data given as mean ± S.E.M. PR: placing response; SRR: surface righting reflex; CA: cliff avoidance; NGR: negative geotaxis reflex; TS: tactile startle; ET: ear twitch; ARR: air righting reflex; OF: open field traversal; WS: wire suspension. Table 1 Neurodevelopmental milestones in Nlgn4−/− and WT mice: Overview of the statistical analysis results obtained with two-way ANOVA (compare figure 1). Test

Readout

Data scale

Two-way ANOVA Male

Physical development

Body weight Eye opening Ear opening

Interval Binary Binary

Neurological reflexes

Placing response Surface righting Negative geotaxis Cliff avoidance Ear twitch Tactile startle Air righting

Binary Interval Interval Interval Binary Binary Binary

Neuromotorcoordination

Open field Wire suspension

Interval Interval

Female

Genotype

Time

Interaction

Genotype

Time

Interaction

0.4375

p < 0.0001

0.6107

0.9406

p < 0.0001

0.6107

0.5361 0.5412 0.6054

p < 0.0001 p < 0.0001 p < 0.0001

0.8166 0.702 0.5686

0.8514 0.9239 0.0679

p < 0.0001 p < 0.0001 p < 0.0001

0.9864 0.236 0.9507

0.4887 0.9883

p < 0.0001 p < 0.0001

0.8035 0.7893

0.1541 0.9953

p < 0.0001 p < 0.0001

0.9239 0.9985

Table 2 USV frequency parameters in Nlgn4−/− and WT mice of both genders. Parameter Pups Males (+/+) Males (−/−) Females (+/+) Females (−/−) Juveniles Males (+/+) Males (−/−) Females (+/+) Females (−/−)

N

PF start

PF end

PF max

19 14 16 12

83.6 83.8 80.8 77.4

± ± ± ±

1.1 1.2 1.1 2.4

81.7 83.4 79.3 75.5

± ± ± ±

1.0 1.1 0.8 1.9

90.0 90.3 90.3 84.7

± ± ± ±

1.1 0.8 1.0 1.8

83.1 84.1 81.1 77.0

± ± ± ±

1.0 1.0 0.8 2.1

0.01 0.01 0.01 0.01

± ± ± ±

0.00 0.00 0.00 0.00

14 15 16 13

82.5 84.7 85.3 81.7

± ± ± ±

1.7 1.5 1.4 2.0

80.6 84.3 83.7 79.7

± ± ± ±

2.3 2.2 1.4 2.9

88.2 91.0 92.4 87.1

± ± ± ±

2.2 2.0 1.6* 2.5*

82.0 84.9 85.3 81.2

± ± ± ±

1.9 1.8 1.4 2.4

0.01 0.01 0.01 0.01

± ± ± ±

0.01 0.00 0.00 0.00

PF = peak frequency, PF max loc = location of PF max. Mean ± SEM. * Significant (p < 0.05) difference between genotypes corrected for multiple testing (Simes correction).

PF mean

PF max loc

A. Ju et al. / Behavioural Brain Research 270 (2014) 159–164

Pups PND8/9

Juveniles PND22/23 400

Number of calls [#]

400

Nlgn4+/+ Nlgn4-/-

300

200

100

100

19

15

Male

17

13

Female

0.03

300

200

0

163

0

17

16

Male

17

13

Female

Total duration of calls [s]

Genotype, p= 0.015 9.0

9.0

7.5

7.5

6.0

6.0

4.5

4.5

3.0

3.0 1.5

1.5 0.0

19

13

Male

17

12

Female

0.0

Mean duration of calls [s]

0.04

0.04

0.03

0.03

0.02

0.02

0.01

0.01

19

13

Male

17

12

Female

Latency to first call [s]

Gender, p= 0.0007 12

0.036

0.00

17

13

Female

0.009

17

16

Male

12 10

8

8

6

6

4

4

0

16

Male

17

13

Female

Genotype, p= 0.034; Gender, p= 0.011

10

2

17

Genotype, p= 0.009

Gender, p= 0.027

0.00

0.028

0.097

2

16

13

Male

14

9

Female

0

15

16

Male

16

12

Female

Fig. 2. Ultrasound vocalization (USV) measures in Nlgn4−/− versus Nlgn4+/+ pups and juvenile mice. The left column presents results for pups, the right for juveniles. Gender and genotype results obtained with ANOVA for the respective time point are written underneath the corresponding panel wherever applicable. Post hoc test results (Student’s t-test) are displayed within the panels. N numbers are given in the columns. Data are presented as mean ± S.E.M.

Most of the parameters measured for the assessment of neurodevelopmental reflex maturation and neuromotor coordination are highly dependent on a regular muscular and motor function or acquisition of symmetrical coordination as transmitted from the inner ear to the central vestibular system located in the hindbrain

and integrated with information from other neural systems [19,20]. A negative geotaxis additionally reflects sensorimotor function, and wire suspension is highly affected by the information from visual and proprioceptive systems [21]. Since Nlgn4 is expressed lowest in cerebellum and no difference in activity or motor performance

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was observed in adult Nlgn4−/− mice [9], the absence of respective effects on neurodevelopment may not be too surprising. As opposed to the normal neurodevelopmental milestones measured in Nlgn4−/− mice, other ASD mouse models, e.g. Mecp2 mutant mice modeling Rett syndrome, revealed abnormal neurodevelopment comprising, e.g. delay of reflex maturation and of wire suspension performance as a neuromotor coordination readout [22]. Whereas Mecp2 influences hundreds of other genes [23,24], inducing a highly complex and severe phenotype upon loss-offunction, the Nlgn4 null mutation is apparently well compensated for regarding many investigated readouts, e.g. synapse number or global levels of synaptic proteins, to just name a few [25]. Nevertheless, the robust communication deficit uncovered by USV monitoring further underlines the autism-relevance of the Nlgn4−/− mouse line.

[4]

[5] [6]

[7]

[8]

[9]

[10]

Conflict of interest None to report.

[11]

Author’s contribution

[12]

A.J. and M.T. performed all behavioral analyses on adult and neonatal mice. K.H., together with A.J., conducted the USV monitoring. A.J., K.H., M.T. and H.E. performed the statistical analyses, designed the figures and interpreted the final data. H.E., supported by D.K. and N.B., planned, supervised and coordinated the project. H.E. and A.J. wrote the manuscript. All authors contributed to the current version of the paper.

[13]

[14] [15]

[16]

Acknowledgments [17]

This work was supported by the Max Planck Society, the Max Planck Förderstiftung, the DFG (CNMPB) as well as by EU-AIMS. The research of EU-AIMS receives support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115300, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013), from the EFPIA companies, and from Autism Speaks.

[18] [19] [20] [21]

[22]

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