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Reduced ultrasound vocalization in NL-4-KO mice. Ultrasonic vocalizations of individual male WT and NL-4-KO mice were measured upon contact of the male test mouse with an unfamiliar female mouse in estrous. ( A ) Frequency spectrogram of typical ultrasonic vocalizations of a male WT mouse. ( B ) Frequency spectrograms of ultrasonic vocalizations of a male WT ( Upper ) and a male NL-4-KO ( Lower ) mouse, indicating the reduced number of vocalizations in NL-4-KOs. ( C ) Quantitative analysis of the latency between the time the female mouse was put into the arena of the male test mouse and the first ultrasonic call of the male in WT ( n ϭ 20) and NL-4-KO ( n ϭ 16) mice. Data are presented as mean Ϯ SEM. The asterisk indicates a significant increase in latency in NL-4-KO mice ( P ϭ 0.03). ( D ) Quantitative analysis of the number of ultrasonic calls made by male WT ( n ϭ 20) and NL-4-KO ( n ϭ 16) mice during a 3-min session. Data are presented as mean Ϯ SEM. The asterisk indicates a significant reduction in the number of calls made by NL-4-KO mice ( P ϭ 0.02).
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Autism spectrum conditions (ASCs) are heritable conditions characterized by impaired reciprocal social interactions, deficits in language acquisition, and repetitive and restricted behaviors and interests. In addition to more complex genetic susceptibilities, even mutation of a single gene can lead to ASC. Several such monogenic heritable ASC forms...
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... acquisition and communication are key symptoms of ASCs. To test whether vocal behavior is impaired in NL-4-KO mice, we recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultra- sound calls of different types (Fig. 3A). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls (P 0.03; Fig. 3C), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls (P 0.02; Fig. 3 B and D). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO ...
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... recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultra- sound calls of different types (Fig. 3A). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls (P 0.03; Fig. 3C), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls (P 0.02; Fig. 3 B and D). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not ...
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... In this setting, male mice produce bouts of frequency-modulated ultra- sound calls of different types (Fig. 3A). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls (P 0.03; Fig. 3C), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls (P 0.02; Fig. 3 B and D). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not shown), and that sensory deficits could be ruled out (SI Figs. 8 and 10A), these results indicate that the NL-4-KOs are either ...
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... and mental retardation (9). The present study shows that an analogous loss-of-function mutation in the orthologous mouse Nlgn4 gene leads to a selective perturbation of social behavior and vocalization. Adult male NL-4-KO mice exhibit a reduced interest in conspecific mice (Fig. 2) and reduced ultrasonic vocalization upon contact with a female (Fig. 3), whereas sensory ability, locomotor and exploratory activity, anxiety behavior, and learning and memory are not detectably altered. Thus, NL-4-KOs specifically and selectively mimic two of the three cardinal symptoms of ASCs in humans, i.e., reduced or aberrant social interactions and impaired communication. In addition, we detected ...
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... learning and memory (hidden platform training in Morris water maze, SI Fig. 10 B and C ; cued and contextual fear conditioning; SI Fig. 10 E ), and cognitive flexibility (reversal training in Morris water maze; SI Fig. 10 D ). In all these assays, NL-4-KOs were indistinguishable from their WT littermates, indicating that the lack of NL-4 does not cause gross sensory deficits or generalized perturbations of behavior. In addition, NL-4-KOs did not exhibit an altered seizure propensity (SI Fig. 11). Because deficits in reciprocal social interactions are a key symptom of ASCs, we next studied NL-4-KO and WT mice in behavioral paradigms designed to specifically assess social behavior. First, direct contacts between pairs of genotypically identical NL-4-KO or WT mice were analyzed in an open arena. We found that NL-4-KOs spent 45% less time in direct contact with each other than WT controls ( P ϭ 0.0005; Fig. 2 A ). Second, mice were tested in a tripartite arena consisting of a central compartment connected to two lateral compartments on either side, one of which contained an unfamiliar mouse in a small wire cage, whereas the other contained an identical but empty wire cage. WT mice showed a clear preference for the compartment containing the unfamiliar mouse. They spent 62% more time in and made 27% more visits to the compartment with the unfa- miliar mouse compared with the compartment with the empty wire cage ( P ϭ 0.0001 and P ϭ 0.04, respectively; Fig. 2 B – D ). In contrast, no such preference was observed in NL-4-KO mice. They spent equal time in and made equally frequent visits to the compartment with the unfamiliar mouse as to that with the empty wire cage, resulting in significantly different ratios of time spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.03; Fig. 2 B – D ). At the end of the first session, each mouse was tested in a second session to quantify social preference for a new stranger. A second, unfamiliar mouse was placed into the previously empty wire cage such that the test mouse had a choice between the first, already explored mouse and the novel unfamiliar mouse. In this setting, WT mice showed a clear preference for the compartment containing the novel unfamiliar mouse. They spent 28% more time in the compartment with the unfamiliar mouse compared with that with the familiar mouse ( P ϭ 0.002; Fig. 2 E and F ). Again, no such preference was detectable with NL-4-KOs, which spent roughly equal time in the two compartments, resulting in a significantly different ratio of times spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.04; Fig. 2 E and F ). Third, we investigated the behavior of NL-4-KO and WT mice in the resident-intruder paradigm. This setting can be used to study aggression of the resident related to territorial behavior. We exposed individually housed resident NL-4-KO or WT mice to an unfamiliar intruder mouse and measured the time spent by the resident until it first attacks the intruder. We found that attack latencies were 28% longer for NL-4-KO residents compared with WT controls ( P ϭ 0.004; Fig. 2 G ) and the proportion of individual mice attacking at all during the test period was reduced by 63% ( P ϭ 0.02; Fig. 2 H ). Finally, in a modified version of the resident-intruder setting using an independent cohort of mice, we focused on the correlation between resident (WT or NL-4-KO) escape and intruder approach behavior in a neutral arena. We detected a significant positive correlation between the number of intruder approaches and the number of resident escapes for WT residents (Spearman correlation, Z ϭ 3.1, P ϭ 0.002), whereas no such correlation was observed if NL-4-KOs were used as residents (Spearman correlation, Z ϭ 0.6; P ϭ 0.6) (Fig. 2 I ), showing that NL-4-KOs respond atypically to the behavior of a conspecific. The above data show that NL-4-KOs have selective deficits in reciprocal social behavior reminiscent of the changes in social interactions typically seen in ASC patients. These changes are unlikely to be due to sensory, locomotor, or memory defects, because NL-4-KOs perform normally in tests of olfactory, visual, and auditory abilities (SI Figs. 8 and 10 A ), locomotor activity and coordination (SI Fig. 9 A , B , D , and E ), and memory (SI Fig. 10). Reduced Vocalization in NL-4-KOs. Deficits in language acquisition and communication are key symptoms of ASCs. To test whether vocal behavior is impaired in NL-4-KO mice, we recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultrasound calls of different types (Fig. 3 A ). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls ( P ϭ 0.03; Fig. 3 C ), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls ( P ϭ 0.02; Fig. 3 B and D ). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not shown), and that sensory deficits could be ruled out (SI Figs. 8 and 10 A ), these results indicate that the NL-4-KOs are either less responsive to the social stimuli eliciting the calling or are otherwise inhibited in their propensity to communicate. Reduced Brain Volume in NL-4-KOs. A consistent finding in his- topathological and structural imaging studies on children with ASCs is an increase in brain volume. However, no such alterations are reliably detectable in adult ASC patients or in patients with NLGN4 mutations (8). In addition, analyses of individual brain regions of ASC patients provided suggestive evidence of neuronal loss in cerebellum, thickened frontal cortices, decreased corpus callosum volume, reduced brainstem size, and other less well characterized morphological alterations (15). To test whether related changes occur upon deletion of NL-4 in mice, we analyzed the volume of the whole brain and selected brain regions in NL-4-KO and WT mice by MRI volumetry (Fig. 4 A ). We observed small but significant reductions in the volume of the total brain (1.5% reduction, P ϭ 0.004), cerebellum (4.1% reduction, P ϭ 0.0005), and brainstem (3.8% reduction, P ϭ 0.01) in NL-4-KOs compared with WT controls, whereas ven- tricular volumes were similar in the two genotypes (Fig. 4 B – E ). These findings are compatible with a selective loss of gray matter in the altered brain regions of NL-4-KOs and congruent with data obtained in adult ASC patients. Loss-of-function mutations in the human NLGN4 gene can cause monogenic heritable autism or Asperger syndrome (8, 9) and mental retardation (9). The present study shows that an analogous loss-of-function mutation in the orthologous mouse Nlgn4 gene leads to a selective perturbation of social behavior and vocalization. Adult male NL-4-KO mice exhibit a reduced interest in conspecific mice (Fig. 2) and reduced ultrasonic vocalization upon contact with a female (Fig. 3), whereas sensory ability, locomotor and exploratory activity, anxiety behavior, and learning and memory are not detectably altered. Thus, NL-4-KOs specifically and selectively mimic two of the three cardinal symptoms of ASCs in humans, i.e., reduced or aberrant social interactions and impaired communication. In addition, we detected reductions in the brain size of adult NL-4-KOs (Fig. 4) that corroborate reports of reduced brain size in adult ASC patients (15). These observations establish the NL-4-KO as a genetic animal model of monogenic heritable ASC. We did not detect abnormal repetitive behavior patterns in NL-4-KO mice, which are typically observed in human ASC patients, indicating that NL-4-KOs do not model all characteristic ASC symptoms. This is not unexpected, because not all human ASC patients with NL-4 mutations exhibit repetitive behavior patterns (8), and many of the more nuanced aspects of ASCs are difficult or even impossible to assess in animal models. In the past, all reports on genetic mouse models of ASCs except one were related to syndromic human genetic diseases frequently associated with ASCs, including Rett syndrome, fragile X syndrome, tuberous sclerosis, and neurofibromatosis type 1. The corresponding mouse models with mutations in MeCP2, FMR1, TSC1/TSC2, or NF1 exhibit certain behavioral and morphological changes that are reminiscent of ASCs (16– 25). The same is true for mice in which a limited number of cortical and hippocampal neurons lack the lipid phosphatase PTEN (26), which is functionally linked to TSC2 and mutated in some individuals with ASC (27, 28). Animal models designed to mimic the contribution of environmental factors such as certain viral infections or drugs to ASCs are less well defined. This is mainly due to pleiotropic effects of the corresponding treat- ments, for example, in a valproic acid rat model of autism (29). The key disadvantage of the syndromic and environmental animal models of ASCs mentioned above is that ASC-related phenotypic changes are invariably associated with multiple ad- ditional neurological and behavioral changes not typically seen in ASCs. The same applies to the corresponding human conditions, which are not even always associated with ASC features. In contrast, patients lacking NL-4 have rather specific ASC symptoms without overt or complex comorbidity (8), and NL- 4-KOs exhibit highly selective ASC-like behavioral deficits (Figs. 2 and 3) and only very mild morphological changes in brain size congruent with respective changes seen in many adult ASC patients (Fig. 4), but otherwise no alterations in brain cytoar- chitecture (data not shown) or behavior (SI Figs. 8–11). Thus, in terms of ASC specificity, the NL-4-KO model is an ideal tool for further studies on the biological basis of ASCs. Our data on ...
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... setting, WT mice showed a clear preference for the compartment containing the novel unfamiliar mouse. They spent 28% more time in the compartment with the unfamiliar mouse compared with that with the familiar mouse ( P ϭ 0.002; Fig. 2 E and F ). Again, no such preference was detectable with NL-4-KOs, which spent roughly equal time in the two compartments, resulting in a significantly different ratio of times spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.04; Fig. 2 E and F ). Third, we investigated the behavior of NL-4-KO and WT mice in the resident-intruder paradigm. This setting can be used to study aggression of the resident related to territorial behavior. We exposed individually housed resident NL-4-KO or WT mice to an unfamiliar intruder mouse and measured the time spent by the resident until it first attacks the intruder. We found that attack latencies were 28% longer for NL-4-KO residents compared with WT controls ( P ϭ 0.004; Fig. 2 G ) and the proportion of individual mice attacking at all during the test period was reduced by 63% ( P ϭ 0.02; Fig. 2 H ). Finally, in a modified version of the resident-intruder setting using an independent cohort of mice, we focused on the correlation between resident (WT or NL-4-KO) escape and intruder approach behavior in a neutral arena. We detected a significant positive correlation between the number of intruder approaches and the number of resident escapes for WT residents (Spearman correlation, Z ϭ 3.1, P ϭ 0.002), whereas no such correlation was observed if NL-4-KOs were used as residents (Spearman correlation, Z ϭ 0.6; P ϭ 0.6) (Fig. 2 I ), showing that NL-4-KOs respond atypically to the behavior of a conspecific. The above data show that NL-4-KOs have selective deficits in reciprocal social behavior reminiscent of the changes in social interactions typically seen in ASC patients. These changes are unlikely to be due to sensory, locomotor, or memory defects, because NL-4-KOs perform normally in tests of olfactory, visual, and auditory abilities (SI Figs. 8 and 10 A ), locomotor activity and coordination (SI Fig. 9 A , B , D , and E ), and memory (SI Fig. 10). Reduced Vocalization in NL-4-KOs. Deficits in language acquisition and communication are key symptoms of ASCs. To test whether vocal behavior is impaired in NL-4-KO mice, we recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultrasound calls of different types (Fig. 3 A ). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls ( P ϭ 0.03; Fig. 3 C ), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls ( P ϭ 0.02; Fig. 3 B and D ). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not shown), and that sensory deficits could be ruled out (SI Figs. 8 and 10 A ), these results indicate that the NL-4-KOs are either less responsive to the social stimuli eliciting the calling or are otherwise inhibited in their propensity to communicate. Reduced Brain Volume in NL-4-KOs. A consistent finding in his- topathological and structural imaging studies on children with ASCs is an increase in brain volume. However, no such alterations are reliably detectable in adult ASC patients or in patients with NLGN4 mutations (8). In addition, analyses of individual brain regions of ASC patients provided suggestive evidence of neuronal loss in cerebellum, thickened frontal cortices, decreased corpus callosum volume, reduced brainstem size, and other less well characterized morphological alterations (15). To test whether related changes occur upon deletion of NL-4 in mice, we analyzed the volume of the whole brain and selected brain regions in NL-4-KO and WT mice by MRI volumetry (Fig. 4 A ). We observed small but significant reductions in the volume of the total brain (1.5% reduction, P ϭ 0.004), cerebellum (4.1% reduction, P ϭ 0.0005), and brainstem (3.8% reduction, P ϭ 0.01) in NL-4-KOs compared with WT controls, whereas ven- tricular volumes were similar in the two genotypes (Fig. 4 B – E ). These findings are compatible with a selective loss of gray matter in the altered brain regions of NL-4-KOs and congruent with data obtained in adult ASC patients. Loss-of-function mutations in the human NLGN4 gene can cause monogenic heritable autism or Asperger syndrome (8, 9) and mental retardation (9). The present study shows that an analogous loss-of-function mutation in the orthologous mouse Nlgn4 gene leads to a selective perturbation of social behavior and vocalization. Adult male NL-4-KO mice exhibit a reduced interest in conspecific mice (Fig. 2) and reduced ultrasonic vocalization upon contact with a female (Fig. 3), whereas sensory ability, locomotor and exploratory activity, anxiety behavior, and learning and memory are not detectably altered. Thus, NL-4-KOs specifically and selectively mimic two of the three cardinal symptoms of ASCs in humans, i.e., reduced or aberrant social interactions and impaired communication. In addition, we detected reductions in the brain size of adult NL-4-KOs (Fig. 4) that corroborate reports of reduced brain size in adult ASC patients (15). These observations establish the NL-4-KO as a genetic animal model of monogenic heritable ASC. We did not detect abnormal repetitive behavior patterns in NL-4-KO mice, which are typically observed in human ASC patients, indicating that NL-4-KOs do not model all characteristic ASC symptoms. This is not unexpected, because not all human ASC patients with NL-4 mutations exhibit repetitive behavior patterns (8), and many of the more nuanced aspects of ASCs are difficult or even impossible to assess in animal models. In the past, all reports on genetic mouse models of ASCs except one were related to syndromic human genetic diseases frequently associated with ASCs, including Rett syndrome, fragile X syndrome, tuberous sclerosis, and neurofibromatosis type 1. The corresponding mouse models with mutations in MeCP2, FMR1, TSC1/TSC2, or NF1 exhibit certain behavioral and morphological changes that are reminiscent of ASCs (16– 25). The same is true for mice in which a limited number of cortical and hippocampal neurons lack the lipid phosphatase PTEN (26), which is functionally linked to TSC2 and mutated in some individuals with ASC (27, 28). Animal models designed to mimic the contribution of environmental factors such as certain viral infections or drugs to ASCs are less well defined. This is mainly due to pleiotropic effects of the corresponding treat- ments, for example, in a valproic acid rat model of autism (29). The key disadvantage of the syndromic and environmental animal models of ASCs mentioned above is that ASC-related phenotypic changes are invariably associated with multiple ad- ditional neurological and behavioral changes not typically seen in ASCs. The same applies to the corresponding human conditions, which are not even always associated with ASC features. In contrast, patients lacking NL-4 have rather specific ASC symptoms without overt or complex comorbidity (8), and NL- 4-KOs exhibit highly selective ASC-like behavioral deficits (Figs. 2 and 3) and only very mild morphological changes in brain size congruent with respective changes seen in many adult ASC patients (Fig. 4), but otherwise no alterations in brain cytoar- chitecture (data not shown) or behavior (SI Figs. 8–11). Thus, in terms of ASC specificity, the NL-4-KO model is an ideal tool for further studies on the biological basis of ASCs. Our data on mutant mice show that the Nlgn4 mutation alone, without the contribution of confounding external factors, causes ASC- related symptoms in mice, and the same is likely to be the case in human ASC patients with NLGN4 mutations. Very recently, the phenotype of NL-3 mutant mice carrying a mutation implicated in monogenic heritable autism (R451C; ref. 8) was described (30). NL-3 R451C mice exhibit deficits in social interaction that are similar to but less selective than the behavioral phenotype of NL-4-KO described here (Fig. 2 A – F ), as NL-3 R451C mice also exhibit other behavioral changes such as enhanced spatial learning, which is not seen in NL-4-KOs and could indirectly affect social behavior. Aggressive behavior and vocalization, which are specifically perturbed in NL-4-KOs (Figs. 2 G – I and 3), were not tested in NL-3 R451C mutant mice. The behavioral changes in NL-3 R451C mutant mice are associated with a selective increase in inhibitory synaptic transmission, indicating that a perturbation of synaptic function may contribute to the behavioral deficits seen in these mice. Currently, no efficient therapies for ASCs are available. However, the fact that neurological symptoms in mutant mouse models of Rett and fragile X syndrome can be reversed (31, 32) indicates that ASC-related conditions might ultimately be cur- able. Whether the NL-4-KO model described here or the NL-3 model described earlier (30) are of general relevance for ASCs in humans and can thus be used systematically for the development of novel diagnostic and therapeutic strategies cannot yet be decided. Mutations in NLGN genes are rare among ASC patients (33–36) and, in several of the identified monogenic heritable ASC cases, the genes involved cannot be linked directly to NL function (5, 6). However, the fact that not only mutations of NLGN4 or NLGN3 but also mutations of NRXN1 or SHANK3 can cause monogenic heritable ASCs indicates that a protein network that regulates the maturation and function of synapses in the brain is at the core of a major ASC susceptibility pathway, which can be studied with unique specificity using the NL-4-KO described here. NLs ...
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... (rota-rod, SI Fig. 9 A ; open field, SI Fig. 9 D ), exploratory behavior (hole board; SI Fig. 9 B ), overall curiosity toward inanimate objects (object preference; SI Fig. 9 C ), anxiety (open field, SI Fig. 9 E ; elevated plus maze, SI Fig. 9 F ), learning and memory (hidden platform training in Morris water maze, SI Fig. 10 B and C ; cued and contextual fear conditioning; SI Fig. 10 E ), and cognitive flexibility (reversal training in Morris water maze; SI Fig. 10 D ). In all these assays, NL-4-KOs were indistinguishable from their WT littermates, indicating that the lack of NL-4 does not cause gross sensory deficits or generalized perturbations of behavior. In addition, NL-4-KOs did not exhibit an altered seizure propensity (SI Fig. 11). Because deficits in reciprocal social interactions are a key symptom of ASCs, we next studied NL-4-KO and WT mice in behavioral paradigms designed to specifically assess social behavior. First, direct contacts between pairs of genotypically identical NL-4-KO or WT mice were analyzed in an open arena. We found that NL-4-KOs spent 45% less time in direct contact with each other than WT controls ( P ϭ 0.0005; Fig. 2 A ). Second, mice were tested in a tripartite arena consisting of a central compartment connected to two lateral compartments on either side, one of which contained an unfamiliar mouse in a small wire cage, whereas the other contained an identical but empty wire cage. WT mice showed a clear preference for the compartment containing the unfamiliar mouse. They spent 62% more time in and made 27% more visits to the compartment with the unfa- miliar mouse compared with the compartment with the empty wire cage ( P ϭ 0.0001 and P ϭ 0.04, respectively; Fig. 2 B – D ). In contrast, no such preference was observed in NL-4-KO mice. They spent equal time in and made equally frequent visits to the compartment with the unfamiliar mouse as to that with the empty wire cage, resulting in significantly different ratios of time spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.03; Fig. 2 B – D ). At the end of the first session, each mouse was tested in a second session to quantify social preference for a new stranger. A second, unfamiliar mouse was placed into the previously empty wire cage such that the test mouse had a choice between the first, already explored mouse and the novel unfamiliar mouse. In this setting, WT mice showed a clear preference for the compartment containing the novel unfamiliar mouse. They spent 28% more time in the compartment with the unfamiliar mouse compared with that with the familiar mouse ( P ϭ 0.002; Fig. 2 E and F ). Again, no such preference was detectable with NL-4-KOs, which spent roughly equal time in the two compartments, resulting in a significantly different ratio of times spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.04; Fig. 2 E and F ). Third, we investigated the behavior of NL-4-KO and WT mice in the resident-intruder paradigm. This setting can be used to study aggression of the resident related to territorial behavior. We exposed individually housed resident NL-4-KO or WT mice to an unfamiliar intruder mouse and measured the time spent by the resident until it first attacks the intruder. We found that attack latencies were 28% longer for NL-4-KO residents compared with WT controls ( P ϭ 0.004; Fig. 2 G ) and the proportion of individual mice attacking at all during the test period was reduced by 63% ( P ϭ 0.02; Fig. 2 H ). Finally, in a modified version of the resident-intruder setting using an independent cohort of mice, we focused on the correlation between resident (WT or NL-4-KO) escape and intruder approach behavior in a neutral arena. We detected a significant positive correlation between the number of intruder approaches and the number of resident escapes for WT residents (Spearman correlation, Z ϭ 3.1, P ϭ 0.002), whereas no such correlation was observed if NL-4-KOs were used as residents (Spearman correlation, Z ϭ 0.6; P ϭ 0.6) (Fig. 2 I ), showing that NL-4-KOs respond atypically to the behavior of a conspecific. The above data show that NL-4-KOs have selective deficits in reciprocal social behavior reminiscent of the changes in social interactions typically seen in ASC patients. These changes are unlikely to be due to sensory, locomotor, or memory defects, because NL-4-KOs perform normally in tests of olfactory, visual, and auditory abilities (SI Figs. 8 and 10 A ), locomotor activity and coordination (SI Fig. 9 A , B , D , and E ), and memory (SI Fig. 10). Reduced Vocalization in NL-4-KOs. Deficits in language acquisition and communication are key symptoms of ASCs. To test whether vocal behavior is impaired in NL-4-KO mice, we recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultrasound calls of different types (Fig. 3 A ). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls ( P ϭ 0.03; Fig. 3 C ), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls ( P ϭ 0.02; Fig. 3 B and D ). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not shown), and that sensory deficits could be ruled out (SI Figs. 8 and 10 A ), these results indicate that the NL-4-KOs are either less responsive to the social stimuli eliciting the calling or are otherwise inhibited in their propensity to communicate. Reduced Brain Volume in NL-4-KOs. A consistent finding in his- topathological and structural imaging studies on children with ASCs is an increase in brain volume. However, no such alterations are reliably detectable in adult ASC patients or in patients with NLGN4 mutations (8). In addition, analyses of individual brain regions of ASC patients provided suggestive evidence of neuronal loss in cerebellum, thickened frontal cortices, decreased corpus callosum volume, reduced brainstem size, and other less well characterized morphological alterations (15). To test whether related changes occur upon deletion of NL-4 in mice, we analyzed the volume of the whole brain and selected brain regions in NL-4-KO and WT mice by MRI volumetry (Fig. 4 A ). We observed small but significant reductions in the volume of the total brain (1.5% reduction, P ϭ 0.004), cerebellum (4.1% reduction, P ϭ 0.0005), and brainstem (3.8% reduction, P ϭ 0.01) in NL-4-KOs compared with WT controls, whereas ven- tricular volumes were similar in the two genotypes (Fig. 4 B – E ). These findings are compatible with a selective loss of gray matter in the altered brain regions of NL-4-KOs and congruent with data obtained in adult ASC patients. Loss-of-function mutations in the human NLGN4 gene can cause monogenic heritable autism or Asperger syndrome (8, 9) and mental retardation (9). The present study shows that an analogous loss-of-function mutation in the orthologous mouse Nlgn4 gene leads to a selective perturbation of social behavior and vocalization. Adult male NL-4-KO mice exhibit a reduced interest in conspecific mice (Fig. 2) and reduced ultrasonic vocalization upon contact with a female (Fig. 3), whereas sensory ability, locomotor and exploratory activity, anxiety behavior, and learning and memory are not detectably altered. Thus, NL-4-KOs specifically and selectively mimic two of the three cardinal symptoms of ASCs in humans, i.e., reduced or aberrant social interactions and impaired communication. In addition, we detected reductions in the brain size of adult NL-4-KOs (Fig. 4) that corroborate reports of reduced brain size in adult ASC patients (15). These observations establish the NL-4-KO as a genetic animal model of monogenic heritable ASC. We did not detect abnormal repetitive behavior patterns in NL-4-KO mice, which are typically observed in human ASC patients, indicating that NL-4-KOs do not model all characteristic ASC symptoms. This is not unexpected, because not all human ASC patients with NL-4 mutations exhibit repetitive behavior patterns (8), and many of the more nuanced aspects of ASCs are difficult or even impossible to assess in animal models. In the past, all reports on genetic mouse models of ASCs except one were related to syndromic human genetic diseases frequently associated with ASCs, including Rett syndrome, fragile X syndrome, tuberous sclerosis, and neurofibromatosis type 1. The corresponding mouse models with mutations in MeCP2, FMR1, TSC1/TSC2, or NF1 exhibit certain behavioral and morphological changes that are reminiscent of ASCs (16– 25). The same is true for mice in which a limited number of cortical and hippocampal neurons lack the lipid phosphatase PTEN (26), which is functionally linked to TSC2 and mutated in some individuals with ASC (27, 28). Animal models designed to mimic the contribution of environmental factors such as certain viral infections or drugs to ASCs are less well defined. This is mainly due to pleiotropic effects of the corresponding treat- ments, for example, in a valproic acid rat model of autism (29). The key disadvantage of the syndromic and environmental animal models of ASCs mentioned above is that ASC-related phenotypic changes are invariably associated with multiple ad- ditional neurological and behavioral changes not typically seen in ASCs. The same applies to the corresponding human conditions, which are not even always associated with ASC features. In contrast, patients lacking NL-4 have rather specific ASC symptoms without overt or complex comorbidity (8), and NL- 4-KOs exhibit highly selective ASC-like behavioral deficits (Figs. 2 and 3) and only very mild morphological changes in brain size congruent with respective changes seen in many adult ASC ...
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... inanimate objects (object preference; SI Fig. 9 C ), anxiety (open field, SI Fig. 9 E ; elevated plus maze, SI Fig. 9 F ), learning and memory (hidden platform training in Morris water maze, SI Fig. 10 B and C ; cued and contextual fear conditioning; SI Fig. 10 E ), and cognitive flexibility (reversal training in Morris water maze; SI Fig. 10 D ). In all these assays, NL-4-KOs were indistinguishable from their WT littermates, indicating that the lack of NL-4 does not cause gross sensory deficits or generalized perturbations of behavior. In addition, NL-4-KOs did not exhibit an altered seizure propensity (SI Fig. 11). Because deficits in reciprocal social interactions are a key symptom of ASCs, we next studied NL-4-KO and WT mice in behavioral paradigms designed to specifically assess social behavior. First, direct contacts between pairs of genotypically identical NL-4-KO or WT mice were analyzed in an open arena. We found that NL-4-KOs spent 45% less time in direct contact with each other than WT controls ( P ϭ 0.0005; Fig. 2 A ). Second, mice were tested in a tripartite arena consisting of a central compartment connected to two lateral compartments on either side, one of which contained an unfamiliar mouse in a small wire cage, whereas the other contained an identical but empty wire cage. WT mice showed a clear preference for the compartment containing the unfamiliar mouse. They spent 62% more time in and made 27% more visits to the compartment with the unfa- miliar mouse compared with the compartment with the empty wire cage ( P ϭ 0.0001 and P ϭ 0.04, respectively; Fig. 2 B – D ). In contrast, no such preference was observed in NL-4-KO mice. They spent equal time in and made equally frequent visits to the compartment with the unfamiliar mouse as to that with the empty wire cage, resulting in significantly different ratios of time spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.03; Fig. 2 B – D ). At the end of the first session, each mouse was tested in a second session to quantify social preference for a new stranger. A second, unfamiliar mouse was placed into the previously empty wire cage such that the test mouse had a choice between the first, already explored mouse and the novel unfamiliar mouse. In this setting, WT mice showed a clear preference for the compartment containing the novel unfamiliar mouse. They spent 28% more time in the compartment with the unfamiliar mouse compared with that with the familiar mouse ( P ϭ 0.002; Fig. 2 E and F ). Again, no such preference was detectable with NL-4-KOs, which spent roughly equal time in the two compartments, resulting in a significantly different ratio of times spent in the two compartments (preference index) compared with WT controls ( P ϭ 0.04; Fig. 2 E and F ). Third, we investigated the behavior of NL-4-KO and WT mice in the resident-intruder paradigm. This setting can be used to study aggression of the resident related to territorial behavior. We exposed individually housed resident NL-4-KO or WT mice to an unfamiliar intruder mouse and measured the time spent by the resident until it first attacks the intruder. We found that attack latencies were 28% longer for NL-4-KO residents compared with WT controls ( P ϭ 0.004; Fig. 2 G ) and the proportion of individual mice attacking at all during the test period was reduced by 63% ( P ϭ 0.02; Fig. 2 H ). Finally, in a modified version of the resident-intruder setting using an independent cohort of mice, we focused on the correlation between resident (WT or NL-4-KO) escape and intruder approach behavior in a neutral arena. We detected a significant positive correlation between the number of intruder approaches and the number of resident escapes for WT residents (Spearman correlation, Z ϭ 3.1, P ϭ 0.002), whereas no such correlation was observed if NL-4-KOs were used as residents (Spearman correlation, Z ϭ 0.6; P ϭ 0.6) (Fig. 2 I ), showing that NL-4-KOs respond atypically to the behavior of a conspecific. The above data show that NL-4-KOs have selective deficits in reciprocal social behavior reminiscent of the changes in social interactions typically seen in ASC patients. These changes are unlikely to be due to sensory, locomotor, or memory defects, because NL-4-KOs perform normally in tests of olfactory, visual, and auditory abilities (SI Figs. 8 and 10 A ), locomotor activity and coordination (SI Fig. 9 A , B , D , and E ), and memory (SI Fig. 10). Reduced Vocalization in NL-4-KOs. Deficits in language acquisition and communication are key symptoms of ASCs. To test whether vocal behavior is impaired in NL-4-KO mice, we recorded ultrasonic vocalizations of individual male NL-4-KO and WT mice upon contact with a female mouse in estrous (14). In this setting, male mice produce bouts of frequency-modulated ultrasound calls of different types (Fig. 3 A ). We found that the latency to start calling was 3.2 times longer in NL-4-KOs compared with WT controls ( P ϭ 0.03; Fig. 3 C ), and the total number of calls per session was reduced by 48% in NL-4-KO mice compared with WT controls ( P ϭ 0.02; Fig. 3 B and D ). Given that the acoustic structure of the vocalizations did not differ between NL-4-KO and WT control mice (data not shown), that ventila- tion frequencies are normal in newborn and adult NL-4-KOs (data not shown), and that sensory deficits could be ruled out (SI Figs. 8 and 10 A ), these results indicate that the NL-4-KOs are either less responsive to the social stimuli eliciting the calling or are otherwise inhibited in their propensity to communicate. Reduced Brain Volume in NL-4-KOs. A consistent finding in his- topathological and structural imaging studies on children with ASCs is an increase in brain volume. However, no such alterations are reliably detectable in adult ASC patients or in patients with NLGN4 mutations (8). In addition, analyses of individual brain regions of ASC patients provided suggestive evidence of neuronal loss in cerebellum, thickened frontal cortices, decreased corpus callosum volume, reduced brainstem size, and other less well characterized morphological alterations (15). To test whether related changes occur upon deletion of NL-4 in mice, we analyzed the volume of the whole brain and selected brain regions in NL-4-KO and WT mice by MRI volumetry (Fig. 4 A ). We observed small but significant reductions in the volume of the total brain (1.5% reduction, P ϭ 0.004), cerebellum (4.1% reduction, P ϭ 0.0005), and brainstem (3.8% reduction, P ϭ 0.01) in NL-4-KOs compared with WT controls, whereas ven- tricular volumes were similar in the two genotypes (Fig. 4 B – E ). These findings are compatible with a selective loss of gray matter in the altered brain regions of NL-4-KOs and congruent with data obtained in adult ASC patients. Loss-of-function mutations in the human NLGN4 gene can cause monogenic heritable autism or Asperger syndrome (8, 9) and mental retardation (9). The present study shows that an analogous loss-of-function mutation in the orthologous mouse Nlgn4 gene leads to a selective perturbation of social behavior and vocalization. Adult male NL-4-KO mice exhibit a reduced interest in conspecific mice (Fig. 2) and reduced ultrasonic vocalization upon contact with a female (Fig. 3), whereas sensory ability, locomotor and exploratory activity, anxiety behavior, and learning and memory are not detectably altered. Thus, NL-4-KOs specifically and selectively mimic two of the three cardinal symptoms of ASCs in humans, i.e., reduced or aberrant social interactions and impaired communication. In addition, we detected reductions in the brain size of adult NL-4-KOs (Fig. 4) that corroborate reports of reduced brain size in adult ASC patients (15). These observations establish the NL-4-KO as a genetic animal model of monogenic heritable ASC. We did not detect abnormal repetitive behavior patterns in NL-4-KO mice, which are typically observed in human ASC patients, indicating that NL-4-KOs do not model all characteristic ASC symptoms. This is not unexpected, because not all human ASC patients with NL-4 mutations exhibit repetitive behavior patterns (8), and many of the more nuanced aspects of ASCs are difficult or even impossible to assess in animal models. In the past, all reports on genetic mouse models of ASCs except one were related to syndromic human genetic diseases frequently associated with ASCs, including Rett syndrome, fragile X syndrome, tuberous sclerosis, and neurofibromatosis type 1. The corresponding mouse models with mutations in MeCP2, FMR1, TSC1/TSC2, or NF1 exhibit certain behavioral and morphological changes that are reminiscent of ASCs (16– 25). The same is true for mice in which a limited number of cortical and hippocampal neurons lack the lipid phosphatase PTEN (26), which is functionally linked to TSC2 and mutated in some individuals with ASC (27, 28). Animal models designed to mimic the contribution of environmental factors such as certain viral infections or drugs to ASCs are less well defined. This is mainly due to pleiotropic effects of the corresponding treat- ments, for example, in a valproic acid rat model of autism (29). The key disadvantage of the syndromic and environmental animal models of ASCs mentioned above is that ASC-related phenotypic changes are invariably associated with multiple ad- ditional neurological and behavioral changes not typically seen in ASCs. The same applies to the corresponding human conditions, which are not even always associated with ASC features. In contrast, patients lacking NL-4 have rather specific ASC symptoms without overt or complex comorbidity (8), and NL- 4-KOs exhibit highly selective ASC-like behavioral deficits (Figs. 2 and 3) and only very mild morphological changes in brain size congruent with respective changes seen in many adult ASC patients (Fig. 4), but otherwise no alterations in brain cytoar- chitecture (data not shown) or behavior (SI Figs. 8–11). Thus, in ...
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The FOXP2 gene is important for the development of proper speech motor control in humans. However, the role of the gene in general vocal behavior in other mammals, including mice, is unclear. Here, we track the vocal development of Foxp2 heterozygous knockout (Foxp2+/−) mice and their wildtype (WT) littermates from juvenile to adult ages, and obser...
Citations
... While macrocephaly is a consistent physical characteristic of children with autism [2,[87][88][89] and was also observed in some ASD-like animal models [90,91], clinical studies have also demonstrated microcephaly among some patients with idiopathic autism [92,93]. The reduction in brain volume was also reported in mouse models with autism-like phenotypes [2], such as nlgn3 KO [94], nlgn4 KO [95] and arx KO mice [96]. In human, mutations of the X-linked ASD related genes encoding neuroligins have been linked to decreased brain volume [97]. ...
Autism Spectrum Disorders (ASD) comprise a range of early age-onset neurodevelopment disorders with genetic heterogeneity. Most ASD related genes are involved in synaptic function, which is regulated by mature brain-derived neurotrophic factor (mBDNF) and its precursor proBDNF in a diametrically opposite manner: proBDNF inhibits while mBDNF potentiates synapses. Here we generated a knock-in mouse line (BDNFmet/leu) in which the conversion of proBDNF to mBDNF is attenuated. Biochemical experiments revealed residual mBDNF but excessive proBDNF in the brain. Similar to other ASD mouse models, the BDNFmet/leu mice showed reduced dendritic arborization, altered spines, and impaired synaptic transmission and plasticity in the hippocampus. They also exhibited ASD-like phenotypes, including stereotypical behaviors and deficits in social interaction. Moreover, the plasma proBDNF/mBDNF ratio was significantly increased in ASD patients compared to normal children in a case-control study. Thus, deficits in proBDNF to mBDNF conversion in the brain may contribute to ASD-like behaviors, and plasma proBDNF/mBDNF ratio may be a potential biomarker for ASD.
... Neuroligins (NLGNs) are a group of postsynaptic cell adhesion molecules. They control the maturity and function of excitatory and inhibitory synapses in the mammalian brain (Jamain et al., 2008;Südhof, 2008). Mutations of NLGN3 and NLGN4 in humans are associated with seizures, X-linked intellectual disability, and autistic behavior. ...
The salient features of autism spectrum disorder (ASD) encompass persistent difficulties in social communication, as well as the presence of restricted and repetitive facets of behavior, hobbies, or pursuits, which are often accompanied with cognitive limitations. Over the past few decades, a sizable number of studies have been conducted to enhance our understanding of the pathophysiology of ASD. Preclinical rat models have proven to be extremely valuable in simulating and analyzing the roles of a wide range of established environmental and genetic factors. Recent research has also demonstrated the significant involvement of the endocannabinoid system (ECS) in the pathogenesis of several neuropsychiatric diseases, including ASD. In fact, the ECS has the potential to regulate a multitude of metabolic and cellular pathways associated with autism, including the immune system. Moreover, the ECS has emerged as a promising target for intervention with high predictive validity. Particularly noteworthy are resent preclinical studies in rodents, which describe the onset of ASD-like symptoms after various genetic or pharmacological interventions targeting the ECS, providing encouraging evidence for further exploration in this area.
... Assessments of social interaction with a novel, same-sex, conspecific as a final measure of novelty exploration have been previously described (Jamain et al., 2008;Morris et al., 2016). As previously described , mice were placed in the corner of the open field chamber (40 × 40 × 35 cm) and allowed to explore and interact with a novel, same-sex conspecific for 10 min. ...
Despite the advent of combination anti‐retroviral therapy (cART), nearly half of people infected with HIV treated with cART still exhibit HIV‐associated neurocognitive disorders (HAND). HAND can be worsened by co‐morbid opioid use disorder. The basal ganglia are particularly vulnerable to HIV‐1 and exhibit higher viral loads and more severe pathology, which can be exacerbated by co‐exposure to opioids. Evidence suggests that dopaminergic neurotransmission is disrupted by HIV exposure, however, little is known about whether co‐exposure to opioids may alter neurotransmitter levels in the striatum and if this in turn influences behavior. Therefore, we assayed motor, anxiety‐like, novelty‐seeking, exploratory, and social behaviors, and levels of monoamines and their metabolites following 2 weeks and 2 months of Tat and/or morphine exposure in transgenic mice. Morphine decreased dopamine levels, but significantly elevated norepinephrine, the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the serotonin metabolite 5‐hydroxyindoleacetic acid, which typically correlated with increased locomotor behavior. The combination of Tat and morphine altered dopamine, DOPAC, and HVA concentrations differently depending on the neurotransmitter/metabolite and duration of exposure but did not affect the numbers of tyrosine hydroxylase‐positive neurons in the mesencephalon. Tat exposure increased the latency to interact with novel conspecifics, but not other novel objects, suggesting the viral protein inhibits exploratory behavior initiation in a context‐dependent manner. By contrast, and consistent with prior findings that opioid misuse can increase novelty‐seeking behavior, morphine exposure increased the time spent exploring a novel environment. Finally, Tat and morphine interacted to affect locomotor activity in a time‐dependent manner, while grip strength and rotarod performance were unaffected. Together, our results provide novel insight into the unique effects of HIV‐1 Tat and morphine on monoamine neurochemistry that may underlie their divergent effects on motor and exploratory behavior.
... Assessments of social interaction with a novel, same-sex, conspecific as a final measure of novelty exploration have been previously described (Jamain et al., 2008;Morris et al., 2016). As previously described , mice were placed in the corner of the open field chamber (40 × 40 × 35 cm) and allowed to explore and interact with a novel, same-sex conspecific for 10 min. ...
Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-β were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.
... Despite these marked changes in tactile reactivity and texture discrimination, Nlgn2 +/mice showed no alterations in anxiety-like or social interaction behaviors compared to control littermates ( Fig. 1d-g and Extended Data Fig. 1a,d,e), although Nlgn2 -/mice exhibited reduced time spent in the center of the open field (Extended Data Fig. 1a,c,d). By contrast, Nlgn4-mutant animals 34 did not exhibit detectable tactile or anxiety-like behavioral alterations (Extended Data Fig. 1f-i), and prior studies observed intact social behaviors in these mutant animals 35 , which may be consistent with significant evolutionary divergence of Nlgn4 in the mouse genome from other mammalian NLGN4 genes 36,37 . ...
... Nlgn4-null mice were obtained from the Mutant Mouse Resource & Research Centers Repository (BayGenomics, 010566-UCD) and were previously described 34 . Avil Cre mice were obtained from F. Wang (Duke University) and were previously described 56 . ...
Altered somatosensory reactivity is frequently observed among individuals with autism spectrum disorders (ASDs). Here, we report that although multiple mouse models of ASD exhibit aberrant somatosensory behaviors in adulthood, some models exhibit altered tactile reactivity as early as embryonic development, whereas in others, altered reactivity emerges later in life. Additionally, tactile overreactivity during neonatal development is associated with anxiety-like behaviors and social behavior deficits in adulthood, whereas tactile overreactivity that emerges later in life is not. The locus of circuit disruption dictates the timing of aberrant tactile behaviors, as altered feedback or presynaptic inhibition of peripheral mechanosensory neurons leads to abnormal tactile reactivity during neonatal development, whereas disruptions in feedforward inhibition in the spinal cord lead to touch reactivity alterations that manifest later in life. Thus, the developmental timing of aberrant touch processing can predict the manifestation of ASD-associated behaviors in mouse models, and differential timing of sensory disturbance onset may contribute to phenotypic diversity across individuals with ASD.
... Many mouse models of autism display impaired USV, including ones that model genetic alterations such as Shank1/2/3, neuroligin-3/4, Tsc2, Fmr1, 15q11-13, 16p11.2 (Jamain et al., 2008;Young et al., 2010;Ju et al., 2014;Wöhr 2014;Kogan et al., 2015;Yang et al., 2015;Brunner et al., 2015;Stoppel et al., 2018;Rotschafer et al., 2012), as well as ones mimicking environmental risks such as maternal immune activation and exposure to valproic acid (Malkova et al., 2012;Cezar et al., 2018). In addition, autism risk genes involved in human speech and language development such as Foxp2, Foxp1 and Cntnap2 also play a role in mouse vocalization, supported by findings in respective transgenic mouse models that display altered USV behavior (Peñagarikano et al., 2011;Brunner et al., 2015;Castellucci et al., 2016;Chabout et al., 2016;Schaafsma et al., 2017;Usui et al., 2017). ...
Background: Deficits in social communication and language development is a hallmark of autism spectrum disorder currently with no cure. Interventional studies using animal models have been very limited in demonstrating improved vocal communication. Autism has been proposed to involve metabolic dysregulation. Ketogenic diet (KD) is a metabolism-based therapy for medically intractable epilepsy, and its applications in other neurological conditions have been increasingly tested. However, how it would affect vocal communication has not been explored. The BTBR mouse strain is considered a model of idiopathic autism. They display robust deficits in vocalization during social interaction, and have metabolic changes implicated in autism.
Methods: We investigated the effects of KD on ultrasonic vocalizations (USVs) in juvenile and adult BTBR mice during male-female social encounters.
Results: After a brief treatment with KD, the amount, spectral bandwidth, and much of the temporal structure of USVs were robustly improved in both juvenile and adult BTBR mice. Composition of call categories and transitioning between individual call subtypes was more effectively improved in juvenile BTBR mice.
Limitations: Although sharing certain attributes, mouse vocalization is unlikely to model all aspects in the development and deficits of human language. KD is highly restrictive and can be difficult to administer, especially for many people with autism who have narrow food selections. Side effects and potential influence on development should also be considered. Future studies are required to tease apart the molecular mechanisms of KD's effects on vocalization.
Conclusions: Together, our data provide further support to the hypothesis that metabolism-based dietary intervention could modify disease expression, including core symptoms, in autism.
... ASD mouse models have shown altered retinal structure and function. The neuroligin-4 knock out (NL4-KO) reduces glycine receptor clusters (GlyRα1) clusters in the inner plexiform layer and the phenotype exhibits ASD features of fewer social interactions and greater delays in communication between littermates compared with wild type mice (Jamain et al., 2008;Hoon et al., 2011). In another model, the offspring of mice exposed to valproic acid (VPA), to induce poor social abilities and heightened anxiety, had an upregulation of the mGLUR5 receptor. ...
The retina and brain share similar neurochemistry and neurodevelopmental origins, with the retina, often viewed as a “window to the brain.” With retinal measures of structure and function becoming easier to obtain in clinical populations there is a growing interest in using retinal findings as potential biomarkers for disorders affecting the central nervous system. Functional retinal biomarkers, such as the electroretinogram, show promise in neurological disorders, despite having limitations imposed by the existence of overlapping genetic markers, clinical traits or the effects of medications that may reduce their specificity in some conditions. This narrative review summarizes the principal functional retinal findings in central nervous system disorders and related mouse models and provides a background to the main excitatory and inhibitory retinal neurotransmitters that have been implicated to explain the visual electrophysiological findings. These changes in retinal neurochemistry may contribute to our understanding of these conditions based on the findings of retinal electrophysiological tests such as the flash, pattern, multifocal electroretinograms, and electro-oculogram. It is likely that future applications of signal analysis and machine learning algorithms will offer new insights into the pathophysiology, classification, and progression of these clinical disorders including autism, attention deficit/hyperactivity disorder, bipolar disorder, schizophrenia, depression, Parkinson’s, and Alzheimer’s disease. New clinical applications of visual electrophysiology to this field may lead to earlier, more accurate diagnoses and better targeted therapeutic interventions benefiting individual patients and clinicians managing these individuals and their families.
... 6,7 However, the role of NLGN1 in disease has been scarce, despite Nlgn1 being a main component of glutamatergic terminals expressed at relatively high levels as compared with other Nlgn family members. [8][9][10] At glutamatergic synapses, Nlgn1 can recruit AMPA (AMPARs) and NMDA receptors (NMDARs) through interactions mediated by the intracellular and extracellular domains, respectively. [11][12][13][14][15] Moreover, it has been J o u r n a l P r e -p r o o f recurrently reported that loss of Nlgn1 by knockout or knockdown approaches produces a reduction in LTP accompanied by major a decrease in NMDARmediated synaptic currents. ...
Alzheimer’s disease (AD) is characterized by memory impairments and age-dependent synapse loss. Experimental and clinical studies have shown decreased expression of the glutamatergic protein Neuroligin-1 (Nlgn1) in AD. However, the consequences of a sustained reduction of Nlgn1 are unknown. Here, we generated a knockin mouse that reproduces the NLGN1 Thr271fs mutation, identified in heterozygosis in a familial case of AD. We found that Nlgn1 Thr271fs mutation abolishes Nlgn1 expression in mouse brain. Importantly, heterozygous Nlgn1 Thr271fs mice showed delay-dependent amnesia for recognition memory. Electrophysiological recordings uncovered age-dependent impairments in basal synaptic transmission and long-term potentiation (LTP) in CA1 hippocampal neurons of heterozygous Nlgn1 Thr271fs mice. In contrast, homozygous Nlgn1 Thr271fs mice showed impaired fear-conditioning memory and normal basal synaptic transmission, suggesting unshared mechanisms for a partial or total loss of Nlgn1. These data suggest that decreased Nlgn1 may contribute to the synaptic and memory deficits in AD.
... Nlgn4, arguably the most enigmatic of the four family members, is expressed at lower levels than the other neuroligins in mice (Varoqueaux et al., 2006), but plays an important role in the regulation of synaptic transmission in different brain regions Delattre et al., 2013;Hammer et al., 2015;Unichenko et al., 2018;Zhang et al., 2018). Strikingly, loss-of-function mutations in the human Nlgn4 ortholog NLGN4X cause autism spectrum disorder (ASD) in humans (Jamain et al., 2003;Yan et al., 2005;Kopp et al., 2021), and Nlgn4 knockout (KO) mice exhibit ASD-like traits (Jamain et al., 2008;El-Kordi et al., 2013;Ju et al., 2014), establishing Nlgn4 KO mice as construct-valid and face-valid ASD models that can yield important insights into the pathomechanisms of ASD. ...
... Mice were housed in individually ventilated cages or in filtertop cages within a ventilated cabinet (Scantainer) at a constant temperature and a 12 h light/dark cycle (lights on at 7:00 A.M.) with food and water available ad libitum. Nlgn4 KO (RRID:MGI:3775814; Jamain et al., 2008) and wild-type (WT) littermates from heterozygote interbreedings were used in all experiments. Genotyping was conducted as previously described (El-Kordi et al., 2013). ...
... While we allowed the slice cultures to mature in vitro for 3 weeks before performing experiments, it is possible that the peak expression levels of Nlgn4 is not yet attained so difference between genotypes may be obscured in the slice cultures. Nlgn4 protein expression in the brain reaches a plateau 3 weeks after birth (Jamain et al., 2008), and Nlgn4 mRNA expression increases from P1 to P18 in the brainstem (Zhang et al., 2018). Thus, the increased paired-pulse inhibition in the dentate gyrus might result from altered network activity that occurs only at later developmental stages. ...
Loss-of-function mutations in neuroligin-4 (Nlgn4), a member of the neuroligin family of postsynaptic adhesion proteins, cause autism spectrum disorder in humans. Nlgn4 knockout (KO) in mice leads to social behavior deficits and complex alterations of synaptic inhibition or excitation, depending on the brain region. In the present work, we comprehensively analyzed synaptic function and plasticity at the cellular and network levels in hippocampal dentate gyrus of Nlgn4 KO mice. Compared with wild-type littermates, adult Nlgn4 KO mice exhibited increased paired-pulse inhibition of dentate granule cell population spikes, but no impairments in excitatory synaptic transmission or short-term and long-term plasticity in vivo In vitro patch-clamp recordings in neonatal organotypic entorhino-hippocampal slice cultures from Nlgn4 KO and wild-type littermates revealed no significant differences in excitatory or inhibitory synaptic transmission, homeostatic synaptic plasticity, and passive electrotonic properties in dentate granule cells, suggesting that the increased inhibition in vivo is the result of altered network activity in the adult Nlgn4 KO. A comparison with prior studies on Nlgn 1-3 knock-out mice reveals that each of the four neuroligins exerts a characteristic effect on both intrinsic cellular and network activity in the dentate gyrus in vivo.
... The excitation/inhibition imbalance hypothesis suggests that levels of excitation and inhibition are abnormal throughout many brain areas for individuals with autism (Rubenstein and Merzenich, 2003;Jamain et al., 2008), but clear markers for this imbalance in the autistic brain are still needed. At the same time, several experimental studies indicate that inhibition levels in the brain may be implicated in variations in both alternation rate and the prevalence of mixed percepts in binocular rivalry (Robertson et al., 2013;Dieter et al., 2017;Spiegel et al., 2019). ...
When the two eyes are presented with highly distinct stimuli, the resulting visual percept generally switches every few seconds between the two monocular images in an irregular fashion, giving rise to a phenomenon known as binocular rivalry. While a host of theoretical studies have explored potential mechanisms for binocular rivalry in the context of evoked model dynamics in response to simple stimuli, here we investigate binocular rivalry directly through complex stimulus reconstructions based on the activity of a two-layer neuronal network model with competing downstream pools driven by disparate monocular stimuli composed of image pixels. To estimate the dynamic percept, we derive a linear input-output mapping rooted in the non-linear network dynamics and iteratively apply compressive sensing techniques for signal recovery. Utilizing a dominance metric, we are able to identify when percept alternations occur and use data collected during each dominance period to generate a sequence of percept reconstructions. We show that despite the approximate nature of the input-output mapping and the significant reduction in neurons downstream relative to stimulus pixels, the dominant monocular image is well-encoded in the network dynamics and improvements are garnered when realistic spatial receptive field structure is incorporated into the feedforward connectivity. Our model demonstrates gamma-distributed dominance durations and well obeys Levelt's four laws for how dominance durations change with stimulus strength, agreeing with key recurring experimental observations often used to benchmark rivalry models. In light of evidence that individuals with autism exhibit relatively slow percept switching in binocular rivalry, we corroborate the ubiquitous hypothesis that autism manifests from reduced inhibition in the brain by systematically probing our model alternation rate across choices of inhibition strength. We exhibit sufficient conditions for producing binocular rivalry in the context of natural scene stimuli, opening a clearer window into the dynamic brain computations that vary with the generated percept and a potential path toward further understanding neurological disorders.
