Sleepless nights and social plights: medial septum GABAergic hyperactivity in a neuroligin 3–deficient autism model

Social deficits represent a core symptom domain of autism spectrum disorder (ASD), which is often comorbid with sleep disturbances. In this issue of the JCI, Sun et al. explored a medial septum (MS) circuit linking these behaviors in a neuroligin 3 conditional knockout model of autism. They identified GABAergic neuron hyperactivity following neuroligin 3 deletion in the MS. This hyperactivity resulted in the inhibition of the downstream preoptic area (POA) and hippocampal CA2 region, resulting in sleep loss and social memory deficits, respectively. Inactivating the hyperactive MS GABA neurons or activating the POA or CA2 rescued the behavioral deficits. Together, these findings deepen our understanding of neural circuits underlying social and sleep deficits in ASD.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by social interaction difficulties, repetitive behaviors, and restricted interests (1). Affecting approximately 1 in 36 children in the United States, ASD is four times more common in males than females (2). The genetic contributions to ASD are substantial; the Simons Foundation Autism Research Initiative gene database cites over 1000 candidate genes, underscoring the importance of ongoing genetic research to fully understand this disorder.

Many of these candidate genes are crucial for synaptic development, signaling, and plasticity (3), suggesting synaptic dysfunction in ASD. Notably, one of these synaptic genes encodes neuroligin 3 (NLG3), a cell-adhesion molecule required to organize synapses and enable efficient neurotransmission (3, 4). Both gain-of-function mutations and deletions in NLGN3 are observed in ASD (5). Studies show that Nlg3-deficient mice have social interaction impairments (6), social memory deficits (7), and enhanced formation of repetitive routines (8), paralleling ASD-like behaviors.

In addition to social difficulties, 50%–80% of individuals with ASD suffer from sleep disorders (1). The relationship between social interaction and sleep is bidirectional; sleep deprivation can cause social withdrawal (9), and social isolation can negatively impact sleep (10). Studies revealed that Nlg3-knockout rats have reduced social interactions and sleep quality (11). Despite these observations, the neural circuitry linking sleep and social behavior in ASD remain unclear. Understanding these connections is crucial for developing targeted ASD interventions addressing both behavioral deficits.

One brain region that may play a key role in ASD is the medial septum (MS). Previous research has implicated the MS in regulating the sleep-wake cycle (12, 13) and modulating social behaviors (14, 15). However, whether the same MS neurons are involved in both behaviors in an Nlg3-knockout model of autism remains unknown. In this issue of the JCI, Sun and colleagues investigated this question, exploring whether conditional knockout (cKO) of Nlg3 in the MS might underlie the sleep and social disturbances seen in ASD (16).

The authors explored the role of MS NLG3 in social behavior and sleep patterns by first conditionally knocking out Nlg3 (Nlg3-cKO) in the MS of mice. Using the social novelty test, they found that Nlg3-cKO mice displayed normal sociability in comparison with controls, but they showed diminished interest in an unfamiliar mouse, indicative of social memory impairments. Additionally, Nlg3-cKO mice exhibited reduced non–rapid eye movement (NREM) and rapid eye movement (REM) sleep, accompanied by increased wakefulness.

To identify neurons active during social interactions and wakefulness, the authors next labeled the MS for c-Fos, a neural activity marker, and Vgat, a marker for the inhibitory neurotransmitter GABA. Nlg3-cKO mice showed higher levels of c-Fos–expressing neurons, with most coexpressing Vgat, indicating hyperactivity of the MS GABAergic neuron population (MS^GABA).

Having identified this neural population, the authors used optrode recordings to monitor MS^GABA activity during social and sleep-wake behaviors. MS^GABA activity decreased when Nlg3-cKO mice approached an unfamiliar mouse, but increased when avoiding a familiar one. Control mice lacked encoding of approach or avoidance, suggesting that this response was specific to Nlg3-cKO mice. Furthermore, Nlg3-cKO mice showed greater MS^GABA baseline activity than controls during wake, NREM, and REM sleep, with increased firing during transitions from sleep to wake. To investigate the causal relationship between MS^GABA activity and altered behavior, the authors optogenetically activated or inactivated MS^GABA neurons. MS^GABA neuron activation in wild-type mice decreased interactions with an unfamiliar mouse, reduced NREM and REM sleep, and increased wakefulness. Conversely, MS^GABA neuron inactivation in Nlg3-cKO mice increased social novelty, sleep, and decreased wakefulness.

The bidirectional relationship between social interactions and sleep was further dissected by testing the effects of sleep deprivation and social isolation in wild-type mice. After six hours of sleep deprivation, mice showed no social novelty preference, decreased Nlg3 mRNA levels, and increased MS^GABA activation, consistent with previous results. Similarly, four weeks of social isolation increased wakefulness, decreased Nlg3 mRNA levels, and increased MS^GABA activation. These results reveal a dual, causal relationship between MS^GABA hyperactivity and social and sleep deficits.

The authors next sought to understand how MS^GABA circuitry affects social and sleep behaviors. One hypothesis is that distinct MS^GABA populations project to separate downstream regions, each regulating specific behaviors. Alternatively, the same MS^GABA neurons might relay identical information to various downstream targets through multiple collaterals. Unraveling these circuit motifs is essential for understanding how the MS orchestrates diverse behaviors.

To identify these MS^GABA projections, the researchers used viral anterograde tracing in Nlg3-cKO mice. They found MS projections to various brain regions, including the preoptic area (POA), which contains sleep-promoting neurons, and the hippocampal CA2 region, which is implicated in social memory. Viral retrograde tracing labeled specific MS neurons that projected to the POA and CA2 region. Interestingly, about 30% of these neurons overlapped, suggesting that some of these MS neurons indeed collateralize and possibly mediate the tight relationship between sleep and social memory.

To establish a functional role for the POA and CA2, the authors optogenetically inhibited POA and CA2 neurons innervated by MS^GABA terminals in wild-type mice. CA2 inhibition impaired social memory without affecting the sleep-wake cycle, while POA inhibition increased wakefulness without impacting sociability or social memory. These findings suggest that MS^GABA hyperactivity inhibits downstream regions, resulting in these behavioral deficits (Figure 1). Finally, the authors tested whether activating POA and CA2 neurons could rescue the behavioral deficits in Nlg3-cKO mice. Activation of CA2 neurons innervated by MS^GABA terminals restored social memory, increasing interactions with a novel mouse. Activation of POA neurons innervated by MS^GABA terminals increased NREM sleep and decreased wakefulness.

Figure 1

MS^GABA neurons regulate social memory and sleep in Nlg3-cKO mice. Nlg3 knockout induces hyperactivity of GABA neurons in the MS. Hyperactivated MS^GABA neurons impair social memory by reducing CA2 activity and induces sleep loss by decreasing POA activity.

Together, this study reveals a role for MS^GABA neurons in regulating social deficits and sleep disturbances through distinct downstream projections. While these findings provide insights about MS^GABA hyperactivity in autism, it raises questions about the broader implications and potential applications of this research.

Sun et al. (16) discovered social memory and sleep impairments following Nlg3 cKO in the MS, uncovering a neural circuit change underlying autism. Still, unanswered questions remain. The optrode recordings revealed that MS^GABA neurons of Nlg3-cKO mice, but not control mice, encoded social approach and avoidance. A key question is how Nlg3-cKO MS^GABA neurons acquired these encoding properties underlying social behavior.

As NLG3 is a critical postsynaptic molecule involved in synapse organization, MS Nlg3 deletion possibly alters synaptic properties of MS^GABA neurons. This effect is likely not limited to GABAergic neurons, as NLG3 is found at excitatory and inhibitory synapses (17). Such changes could influence multiple synapses, impacting properties like synaptic plasticity. For example, previous studies have shown a loss of cerebellar long-term depression in Nlg3-knockout mice (18) and enhanced hippocampal long-term potentiation in Nlg3-mutant mice (19).

Perhaps NLG3-driven changes in synaptic plasticity affect the encoding capabilities of MS^GABA neurons. A recent study has suggested that hippocampal long-term potentiation enables neurons to encode reward and novelty (20). This finding could explain why Nlg3-CKO mice, but not controls, encoded approach and avoidance. However, it remains unclear why changes to MS synapses in particular affect behavior. The MS mediates various arousal-based behaviors (21–23), so perhaps this region monitors overall internal state changes. Additional studies should investigate how Nlg3 cKO affects MS synaptic plasticity and approach and avoidance computations.

These discoveries open several avenues for future research. Sun et al. (16) link social memory and sleep impairments to Nlg3 cKO in the MS. However, the mechanism by which Nlg3 cKO induces MS^GABA hyperactivity remains an open question. Previous studies have indicated that NLG3 knockdown reduces excitatory synaptic strength (24), increases inhibitory and decreases excitatory miniature events (19), and disrupts tonic endocannabinoid signaling, enhancing GABA release probability (25). While these changes may contribute to MS^GABA hyperactivity, further research should further probe the involved mechanisms. Similarly, whether these observed effects vary by sex, persist throughout the lifespan, or are modulated by hormonal or genetic factors remain to be studied. Ultimately, the Sun et al. findings open doors for exploring targeted pharmaceutical interventions that could mitigate deficits in social memory and sleep in ASD.

RRP was supported by an NIH/National Institute on Alcohol Abuse and Alcoholism K99/R00 Pathway to Independence Award (AA029180).

Address correspondence to: Reesha R. Patel, Address: 320 E. Superior St., Chicago, Illinois 60611, USA. Phone: 317.460.6620; Email: reesha.patel@northwestern.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Copyright: © 2024, Cho et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License.

Reference information: J Clin Invest. 2024;134(19):e184795. https://doi.org/10.1172/JCI184795.

See the related article at Autism-associated neuroligin 3 deficiency in medial septum causes social deficits and sleep loss in mice.

1. Lai MC, et al. Autism. Lancet. 2014;383(9920):896–910.
   View this article via: CrossRef PubMed Google Scholar
2. Maenner MJ. Prevalence and characteristics of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2020. MMWR Surveill Summ. 2023;72(2):1–14.
   View this article via: CrossRef PubMed Google Scholar
3. Guang S, et al. Synaptopathology involved in autism spectrum disorder. Front Cell Neurosci. 2018;12:470.
   View this article via: CrossRef PubMed Google Scholar
4. Südhof TC. Synaptic neurexin complexes: a molecular code for the logic of neural circuits. Cell. 2017;171(4):745–769.
   View this article via: CrossRef PubMed Google Scholar
5. Levy D, et al. Rare de novo and transmitted copy-number variation in autistic spectrum disorders. Neuron. 2011;70(5):886–897.
   View this article via: CrossRef PubMed Google Scholar
6. Modi B, et al. Possible Implication of the CA2 hippocampal circuit in social cognition deficits observed in the neuroligin 3 knock-out mouse, a non-syndromic animal model of autism. Front Psychiatry. 2019;10:513.
   View this article via: CrossRef PubMed Google Scholar
7. Radyushkin K, et al. Neuroligin-3-deficient mice: model of a monogenic heritable form of autism with an olfactory deficit. Genes Brain Behav. 2009;8(4):416–425.
   View this article via: CrossRef PubMed Google Scholar
8. Rothwell PE, et al. Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors. Cell. 2014;158(1):198–212.
   View this article via: CrossRef PubMed Google Scholar
9. Ben Simon E, Walker MP. Sleep loss causes social withdrawal and loneliness. Nat Commun. 2018;9(1):3146.
   View this article via: CrossRef PubMed Google Scholar
10. Kaushal N, et al. Socially isolated mice exhibit a blunted homeostatic sleep response to acute sleep deprivation compared to socially paired mice. Brain Res. 2012;1454:65–79.
    View this article via: CrossRef PubMed Google Scholar
11. Thomas AM, et al. Sleep/wake physiology and quantitative electroencephalogram analysis of the neuroligin-3 knockout rat model of autism spectrum disorder. Sleep. 2017;40(10)
    View this article via: CrossRef PubMed Google Scholar
12. An S, et al. Medial septum glutamatergic neurons control wakefulness through a septo-hypothalamic circuit. Curr Biol. 2021;31(7):1379–1392.
    View this article via: CrossRef PubMed Google Scholar
13. Srividya R, et al. Sleep changes produced by destruction of medial septal neurons in rats. Neuroreport. 2004;15(11):1831–1835.
    View this article via: CrossRef PubMed Google Scholar
14. Wu X, et al. 5-HT modulation of a medial septal circuit tunes social memory stability. Nature. 2021;599(7883):96–101.
    View this article via: CrossRef PubMed Google Scholar
15. Pimpinella D, et al. Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition. Elife. 2021;10:e65580.
    View this article via: CrossRef PubMed Google Scholar
16. Sun H, et al. Autism-associated neuroligin 3 deficiency in medial septum causes social deficits and sleep loss in mice. J Clin Invest. 2024;134(19):e176770.
    View this article via: JCI PubMed CrossRef Google Scholar
17. Budreck EC, Scheiffele P. Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses. Eur J Neurosci. 2007;26(7):1738–1748.
    View this article via: CrossRef PubMed Google Scholar
18. Baudouin SJ, et al. Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism. Science. 2012;338(6103):128–132.
    View this article via: CrossRef PubMed Google Scholar
19. Etherton M, et al. Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function. Proc Natl Acad Sci U S A. 2011;108(33):13764–13769.
    View this article via: CrossRef PubMed Google Scholar
20. Kaganovsky K, et al. Dissociating encoding of memory and salience by manipulating long-term synaptic potentiation [preprint]. https://doi.org/10.1101/2022.01.04.474865 Posted on bioRxiv January 4, 2022.
21. Gangadharan G, et al. Medial septal GABAergic projection neurons promote object exploration behavior and type 2 theta rhythm. Proc Natl Acad Sci U S A. 2016;113(23):6550–6555.
    View this article via: CrossRef PubMed Google Scholar
22. Sweeney P, et al. Appetite suppressive role of medial septal glutamatergic neurons. Proc Natl Acad Sci U S A. 2017;114(52):13816–13821.
    View this article via: CrossRef PubMed Google Scholar
23. Bott JB, et al. Medial septum glutamate neurons are essential for spatial goal-directed memory [preprint]. https://doi.org/10.1101/2022.03.16.484657 Posted on bioRxiv March 17, 2022.
24. Wang L, et al. Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism [published online October 24, 2021]. Mol Psychiatry. https://doi.org/10.1038/s41380-022-01834-x.
25. Földy C, et al. Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling. Neuron. 2013;78(3):498–509.
    View this article via: CrossRef PubMed Google Scholar