Daily oscillation of the excitation/inhibition ratio is disrupted in two mouse models of autism
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eLife assessment
This study presents an important finding on the cause of the widely reported disruption of the excitation to inhibition (E-I) ratio change in Autism Spectrum Disorder (ASD) mouse models. The evidence supporting the main conclusions is solid and well-sampled. These results can be a starting point for studies that assess the functional role of daily oscillations of the E-I ratio in the pathophysiology of ASD, and possibly, reshape our understanding of the nature of the E/I balance alterations that contribute to normal and diseased circuits.
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Abstract
Alterations to the balance between excitation and inhibition (E/I ratio) are postulated to underlie behavioral phenotypes in autism spectrum disorder (ASD) patients and mouse models. However, in wild type mice the E/I ratio is not constant, but instead oscillates across the 24h day. Therefore, we tested whether the E/I oscillation, rather than the overall E/I ratio, is disrupted in two ASD-related mouse lines: Fmr1 KO and BTBR, models of syndromic and idiopathic ASD, respectively. The E/I ratio is dysregulated in both models, but in different ways: the oscillation is lost in Fmr1 KO and reversed in BTBR mice. In both models these phenotypes associate with differences the timing of excitatory and inhibitory synaptic transmission and endocannabinoid signaling compared to wild type mice, but not with altered sleep. These findings raise the possibility that ASD-related phenotypes may be produced by a mismatch of E/I to the appropriate behavioral state, rather than alterations to overall E/I levels per se .
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eLife assessment
This study presents an important finding on the cause of the widely reported disruption of the excitation to inhibition (E-I) ratio change in Autism Spectrum Disorder (ASD) mouse models. The evidence supporting the main conclusions is solid and well-sampled. These results can be a starting point for studies that assess the functional role of daily oscillations of the E-I ratio in the pathophysiology of ASD, and possibly, reshape our understanding of the nature of the E/I balance alterations that contribute to normal and diseased circuits.
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Reviewer #1 (Public Review):
The authors demonstrate that modest oscillatory changes in the E-I ratio occur throughout the day and are linked to changes in both synaptic excitation and inhibition. These conclusions were based on adequately sampled electrophysiological data of stimulation-driven and non-evoked excitatory and inhibitory currents. For these studies, a fixed stimulation was not used across slice recordings but was limited to intensities where the E-I ratio was stable. Two points may need further clarification in the text. Firstly, authors might comment on whether current magnitudes plateau at these stimulation intensities. Secondly, make clear why the cause of E-I balance changes was not elucidated from convergent, evoked measurements in the same cell, but instead relied on non-evoked measures of spontaneous miniature …
Reviewer #1 (Public Review):
The authors demonstrate that modest oscillatory changes in the E-I ratio occur throughout the day and are linked to changes in both synaptic excitation and inhibition. These conclusions were based on adequately sampled electrophysiological data of stimulation-driven and non-evoked excitatory and inhibitory currents. For these studies, a fixed stimulation was not used across slice recordings but was limited to intensities where the E-I ratio was stable. Two points may need further clarification in the text. Firstly, authors might comment on whether current magnitudes plateau at these stimulation intensities. Secondly, make clear why the cause of E-I balance changes was not elucidated from convergent, evoked measurements in the same cell, but instead relied on non-evoked measures of spontaneous miniature excitatory postsynaptic currents and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs) that were recorded separately in different cell populations. mEPSCs and mIPSCs data analysis relied on statistical scrutiny within genotype and could gain additional rigor and benefits to study reproducibility by applying tests ( e.g. two- way repeated measures (RM) ANOVA) that consider the influence of both genotype and time of day. With this approach, the authors could determine in figures 3 and 4 whether control (B6) mice exhibit the predicted increase in mEPSCs and reduction in mIPSCs at ZT0 when compared to its ASD mouse model. In a noteworthy experiment, the authors connect abnormalities in inhibitory oscillations to altered endocannabinoid signaling using measurements of spontaneous (s) IPSCs, where changes in sIPSC charge were noted. The measurements used to make the paper's conclusion lacked consistency and the authors can bridge these differences by testing whether WIN agonist treatment can restore normal daily E/I oscillation in FMR1 KO and BTBR mice using the stimulation-evoked measurements from figure 1. The study used male and female BTBR mice and only male Fmr1 KO mice. Sex- effects in the study were not disclosed, so it is unclear whether daily E/I oscillation changes were similar in male and female BTBR mice or occur at all in female Fmr1 KO mice. Lastly, numerous studies have noted significant changes in the magnitude of the E-I ratio in an autism mouse model and causally linked these changes to alterations in disorder-related behavior or homeostatic regulation of circuit activity. However, in this current study, neither the loss nor reversal of daily E/I ratio oscillation were causally linked to alterations in sleep timing and architecture or any change in behavioral phenotype. On a promising note, the authors did find a slight decrease in NREM delta power in the Fmr1 KO and a larger decrease in the BTBR mice. Future mechanistic studies on this topic may aim to buttress support for E/I oscillations rather than alterations to the overall E/I level in causing autism-related phenotypes by providing supporting examples of biological significance.
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Reviewer #2 (Public Review):
In Bridi et al the authors convincingly show alteration of the E/I ratio oscillation in two mouse models (Fmr1 and BTBR) of ASD. They go on to examine two possible mechanisms that may underlie these changes, 1) sleep/wake cycle and timing and 2) eCB signaling, both of which have been shown to change E/I ratio oscillations. They find that eCB signaling is altered in both models while sleep/wake timing and cycle are normal, concluding that dysfunctional eCB signaling is likely contributing to the changes in E/I oscillation. The experiments are extremely well done, and conclusions are mostly supported by the data, however, there are some concerns with the interpretation of their findings which I will detail below.
- The authors describe the changes in E/I ratio that they observe in the BTBR mouse line as a …
Reviewer #2 (Public Review):
In Bridi et al the authors convincingly show alteration of the E/I ratio oscillation in two mouse models (Fmr1 and BTBR) of ASD. They go on to examine two possible mechanisms that may underlie these changes, 1) sleep/wake cycle and timing and 2) eCB signaling, both of which have been shown to change E/I ratio oscillations. They find that eCB signaling is altered in both models while sleep/wake timing and cycle are normal, concluding that dysfunctional eCB signaling is likely contributing to the changes in E/I oscillation. The experiments are extremely well done, and conclusions are mostly supported by the data, however, there are some concerns with the interpretation of their findings which I will detail below.
- The authors describe the changes in E/I ratio that they observe in the BTBR mouse line as a "phase-shift". However, to actually show a true phase shift they should record at all of the same time points as they did in the Fmr1 model. Based on just two time points the authors have not shown a "Phase-shift" a phase shift would have to show that the other two time points (Z6 and Z18) follow the predicted (-6hr?) shift. These data would also help define the length of the shift.
- Are the changes in E/I ratio presynaptic or postsynaptic? The authors seem to suggest that the synaptic changes they observe are a loss or gain in synapses. Mini-analysis alone is not sufficient for this conclusion. Even if the authors have shown in a previous paper that PPR is unchanged in control mice, presynaptic effects could be contributing to the observed changes in the mouse models studied here. As eCB signaling is thought to be primarily presynaptic this lends additional motivation to explore presynaptic contributions to the observed phenotypes.
- The authors do not make any comparisons between control and ASD model mice at any of their time points. It would be helpful to have additional comparisons between ASD model and control at each time point tested in Fig 1 to relate back to previously published studies that mostly record in the animals' light phase. In other words, please clarify at which phases the ASD E/I ratio is different from the control.
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Reviewer #3 (Public Review):
The authors previously reported a daily oscillation of the excitation/inhibition ratio occurs normally in layer 2/3 cortical neurons in wild-type mice. In this manuscript, they examined the E/I ratio in the primary visual cortex in two different autism mouse models and showed that the daily oscillation was disrupted in both, albeit in different ways. They further demonstrated that complementary changes in excitatory and inhibitory synaptic transmissions were underlying the disrupted E/I ratio, which is also accompanied by alterations in the endocannabinoid signaling but not sleep time in general.
Disruption of the E/I ratio (or balance) has been a major theme of proposed mechanisms underlying sensory and behavioral abnormalities observed in autism spectrum disorder patients and animal models. The …
Reviewer #3 (Public Review):
The authors previously reported a daily oscillation of the excitation/inhibition ratio occurs normally in layer 2/3 cortical neurons in wild-type mice. In this manuscript, they examined the E/I ratio in the primary visual cortex in two different autism mouse models and showed that the daily oscillation was disrupted in both, albeit in different ways. They further demonstrated that complementary changes in excitatory and inhibitory synaptic transmissions were underlying the disrupted E/I ratio, which is also accompanied by alterations in the endocannabinoid signaling but not sleep time in general.
Disruption of the E/I ratio (or balance) has been a major theme of proposed mechanisms underlying sensory and behavioral abnormalities observed in autism spectrum disorder patients and animal models. The demonstration and characterization of the shift/flattening of the daily oscillation of E/I in the two mouse models provide strong evidence for a disruption of the daily dynamic regulation of the E/I ratio instead of an overall change in the absolute level of E/I, at least in layer 2/3 pyramidal neurons in the visual cortex examined here. These results call for a re-visit of previous studies and offer a potential explanation to reconcile conflicting prior reports regarding the valence of E/I ratio changes in different autism models and brain areas, taking the recording time during the day into consideration. It also raises the question of how the dysregulated daily E/I oscillation affects brain functions. On the other hand, the dissociation of sleep and E/I oscillation observed in the autism models may also provide an opportunity to better understand the functional relevance of sleep-dependent E/I oscillation in a normal brain in the future.
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