We show that early-life immune activation caused long-lasting alterations in multiple physiological end points. These end points provide insight on abnormalities (eg, sleep disturbances, altered levels of general activity, seizures) that are often seen in ASD. The fact that the types of perinatal manipulations that produce the core behavioral signs of ASD (Custodio et al, 2017; Malkova et al, 2012) also produce common comorbid physiological disturbances increases confidence in the general approach as a model for ASD, and opens new avenues of research focusing on the pathophysiology and treatment of an immune subtype of this condition. Although more research is necessary to determine in detail how these abnormalities relate to the core features of ASD as measured in mice, our preliminary data (Missig et al, 2016) indicate similar trends in end points such as social interaction.

At the first time point we examined (7 weeks of age), prenatal Poly I:C alone had few effects on its own, although it increased activity during the light-phase. In contrast, postnatal LPS produced more robust effects, including the appearance of SWDs (which were extremely rare in control mice and in Poly I:C alone conditions), light-phase increases in SWS together with corresponding decreases in wakefulness, and dark-phase reductions in activity. The SWDs occurred throughout the day, but were most prevalent during the light phase, when mice are more likely to be sleeping. In general, the ‘two-hit’ (Poly I:C plus LPS) combined treatment produced the strongest effects on all of the metrics studied: in some cases the effects were only nominally different from those of LPS alone (eg, for SWS and SWDs), whereas in other cases statistically significant effects were seen only after combined treatment (eg, effects on spectral power). It is possible that we encountered ‘ceiling effects’ with some of these end points, such that seeing larger effects with the ‘two-hit’ approach was simply not possible under our conditions. It is also possible that using a lower dose of LPS would have produced more modest effects on its own, enabling detection of more pronounced effects of the combined treatment. In preparing for these studies, we performed thorough dose-effect studies to identify an LPS dose that was high enough to produce an immune response in the 9-day old pups, while also being low enough to avoid causing sepsis and lethality. Despite these efforts, two pups died in the days following LPS administration (2 of 37, 5.5%) in the present studies. The dose-effect function was steep, and the dose tested here produced the most reliable effects across various lots of LPS, which can differ in potency. In addition, there was a nominal decrease in the average litter size following Poly I:C that did not reach statistical significance (Vehicle: 6.8 pups/litter vs Poly IC: 5.3 pups/litter, t(10)=1.37, P=0.2). Regardless, our findings provide evidence that the ‘two-hit’ approach produces stronger changes in these end points than either of the individual treatments alone.

Many—though not all—of the effects of perinatal immune activation on these end points were long-lasting. Upon re-analysis (at 12 weeks of age), several of these effects remained evident, including the increases in SWS and appearance of SWDs in LPS-treated mice, and ‘two-hit’ effects on subcutaneous temperature and Gamma power. Several of these changes evolved over time: while the main effect of LPS on SWS and wakefulness persisted, the largest changes were seen in the LPS alone rather than the ‘two-hit’ group. Similarly, while the main effect of LPS on SWDs persisted, differences between LPS alone and the ‘two-hit’ treatment diminished. Only the effects of the ‘two-hit’ treatment on Gamma power during wakefulness and Alpha power during PS persisted, with new significant increases in Beta power during PS emerging over time. In contrast, the pattern of locomotor activity among treatment groups was not consistent between weeks 7 and 12. The reasons for these alterations in patterns are not clear, but obvious possibilities include a gradual reduction of any lingering effects of the acute inflammatory effects of the treatments themselves, the development of homeostatic counteradaptations in response to the induction of persistent changes in brain function, or more general effects of aging. Despite the changes in patterns over time, alterations in EEG power bands may reflect early-life inflammation induced changes in cortical synchrony.

Sleep dysregulation occurs frequently in ASD. The most commonly reported sleep problems are related to insomnia, including difficulties with sleep onset, maintenance, and duration (Richdale and Schreck, 2009). In two genetic rodent models of ASD (16p11.2 or CNTNAP2 deletion), mutant mice show increases in time awake together with either a consolidation or fragmentation of wakefulness (Angelakos et al, 2017; Thomas et al, 2017). These previous findings suggest that the increases in sleep that we observed following early-life immune activation reflect unique consequences of heightened neuroinflammatory processes. Indeed, it has been proposed that increased sleep is a stereotypic response to systemic inflammation, considering evidence that numerous cytokines promote sleep (Krueger et al, 2011). As examples, central or systemic administration of tumor necrosis factor alpha or Interleukin-1 beta increase SWS in a variety of species including mice, rats, primates, and humans (Krueger, 2008). Viewed in aggregate, many of the changes in the ‘two-hit’ model resemble sickness behavior (Bilbo and Schwarz, 2012), including decreased activity, increased SWS, and altered temperature patterns. The presence of these signs may help to differentiate different ASD subtypes.

Although there is considerable evidence for comorbidities of ASD with seizures in humans, corresponding evidence from laboratory animals is currently limited. Seizure disorders are more prevalent in ASD compared with the general population, with estimates of comorbidities as high as 25–33% (Canitano, 2007). Although this is a broad association—with no one particular epilepsy syndrome or type being exclusively associated with ASD—focal seizures are often reported (El Achkar and Spence, 2015). Importantly, epileptiform activity may be a key feature of ASD even in the absence of observable clinical seizures: in an EEG study of 889 ASD patients with no diagnosis of clinical epilepsy, 60% had abnormal epileptiform activity during sleep (Chez et al, 2006). It has been proposed that the co-occurrence of ASD and seizure disorders might result from common pathophysiologies, such as an alterations in excitatory/inhibitory balance (Buckley and Holmes, 2016). Indeed, lower seizure thresholds and/or higher numbers of spontaneous seizures are observed in several genetic mouse models of ASD (Stafstrom and Benke, 2015; Thomas et al, 2017). The epileptiform activity seen in the present studies resembles SWDs seen in EEG recordings from rodent models of absence seizure disorder, and the lack of EMG activity is similar to the behavioral arrest often observed during SWDs (Pearce et al, 2014). We also observed an increase in high-amplitude spikes in the LPS-treated mice. We acknowledge that this spike measure is automated and represents an inherently (but uncontrollably) noisy end point that may also include non-epileptiform activity, as evident by its occasional presence in the control mice. It is conceivable that the increase in spikes in PL mice could reflect, at least in part, increases in interictal spike activity or changes in the intrinsic properties of the EEG signals from these mice. Consistent with the interpretation that these changes are meaningful, however increases in spike was correlated with higher levels of SWDs. In addition, seizures have been previously associated with inflammatory mechanisms: early postnatal LPS administration in rats lowers thresholds for chemically induced seizures (Galic et al, 2008). Our studies provide new evidence that early-life immune system activation can result in spontaneous epileptiform activity, supporting a link between epilepsy and immune dysregulation.

Similar to the shifts in EEG spectral power observed in the current study, abnormal resting-state EEG power has been reported in EEG studies of humans with ASD. Although results have been variable, it has been proposed that the alteration in power structure in ASD is U-shaped, with increased power in lower frequencies (Delta, Theta), decreased middle frequencies (Alpha), and increased in high frequencies (Beta, Gamma) (Wang et al, 2013). The PL-treated mice in the present study approximate half of this shift, at low and middle frequencies, with the changes at high frequencies being inconsistent with this previous model. However, there are discrepancies within the literature, with reports of either increased or decreased Gamma power in ASD (Rojas and Wilson, 2014). Whether the heterogeneity in these findings reflects subtypes of ASD with different etiologies requires additional research. The origin and function of EEG oscillations remains unclear, although it is likely that they represent synchronous postsynaptic potentials resulting from complex neural circuit dynamics (Cohen, 2017). Individual components of EEG oscillation could have particular functions; for example, Alpha oscillations are thought to act as a functional inhibition, dependent on interneuron GABAergic feedback mechanisms (Jensen and Mazaheri, 2010). As such, the decreases in Alpha bands in the present studies may reflect impairment or imbalance of inhibitory feedback. Altered EEG oscillations could have utility as a biomarker that reflects underlying pathological changes in neural circuits in ASD and other neuropsychiatric conditions.

The present findings provide justification for further work. We focused the present studies on male mice, considering the higher prevalence of ASD in males (Werling and Geschwind, 2013), but studies in females may provide insight on whether there are sex differences in sensitivity to these treatments or the severity of the effects. We gave prenatal Poly I:C and postnatal LPS treatment because of evidence showing that TLR3 expression is high but steadily decreasing during prenatal development, whereas TLR4 expression is low but steadily increasing over the same period (Barak et al, 2014). Postnatal administration of LPS also models the high risk of exposure to bacterial infections that occurs during this period of development. PND9 in a mouse approximates neurodevelopmental milestones, including brain growth, gliogenesis, and increases in axonal and dendritic density that are present in humans at full-term birth (Semple et al, 2013). In humans, labor produces over a 10-fold increase of microbial invasion of the amniotic cavity (MIAC) (Romero et al, 2006; Seong et al, 2008), and pre-term birth is highly associated with MIAC and is a risk factor for ASD (Kuzniewicz et al, 2014; Romero et al, 2006). Furthermore, neonates are at high risk for bacterial infections, with an estimated incidence risk of 7.6% for severe infections (Seale et al, 2014). More work is needed to determine if the same pattern of effects would be seen with other combinations of treatments (prenatal LPS followed by postnatal Poly I:C, or two consecutive hits with Poly I:C and/or LPS). Similarly, while we show here that two environmental (immune) manipulations can produce these effects, future work might also examine whether ‘two-hit’ approaches that involve gene × environment interactions can produce comparable outcomes. We have recently used a similar approach to examine sleep and spectral power characteristics during a chronic social defeat stress (CSDS) regimen (Wells et al, 2017). CSDS resulted in a flattening of circadian rhythms, increases in PS and SWS, and alterations in EEG power bands. Although there are some between-study differences in the experimental design and analyses, the effects of CSDS and perinatal immune activation appear distinguishable on numerous levels (eg, effects on PS), suggesting different pathological mechanisms and highlighting the ways in which these various end points are uncoupled and free to vary independently. Our CSDS study highlights the utility of this EEG system for ‘real-time’ studies, to examine these end points before, during, and after an experimental manipulation. In comparison, this current study demonstrates the suitability of this system for evaluating changes in baseline that occur over long periods of time.

A ‘multi-hit’ theory was originally proposed in the context of understanding the causes of cancer (Knudson, 1971) and may provide a useful heuristic for understanding how immune system activation might increase ASD risk in some cases but not others (McDougle et al, 2015). Here, we show that a carefully timed sequence of two environmental hits against a stable genetic background can produce persistent physiological disturbances that often accompany the core signs of ASD. The combined Poly I:C plus LPS treatment produced the largest effects on EEG spectral power. Collapsing the effects over all phases of the sleep-wake continuum yields a potential ‘EEG signature’ of immune-related ASD, with elevations in low power bands and reductions in high power bands. The fact that only some elements of this signature remained evident at the extended (12-week) time point raises the possibility that developmental- or age-related factors can play a role in these metrics. New screening methodologies that identify patients with probable immune-related ASD would make it possible to determine if similar EEG signatures exist in humans, differentiating it from other subtypes of ASD (Wang et al, 2013). This type of advance would facilitate the diagnosis of the condition and the development of more targeted treatments for the various subtypes of ASD, as well as conditions that share common signs (eg, seizure disorders).