Experience-dependent weakening of callosal synaptic connections in the absence of postsynaptic FMRP

  1. Zhe Zhang
  2. Jay R Gibson
  3. Kimberly M Huber

  • Curated by eLife

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    Evaluation Summary:

    The authors find that long-range interhemispheric synapses are selectively weakened following loss of function of the gene mediating fragile X syndrome, the most common inherited form of intellectual disability. Using clever genetic and physiological approaches in mice, the authors show that the effect is cell autonomous and occurs postnatally by impeding the normal developmental strengthening of these synapses. The results convincingly enhance our understanding of the complex pathophysiology of neurological dysfunction in this developmental disorder.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Reduced structural and functional interhemispheric connectivity correlates with the severity of Autism Spectrum Disorder (ASD) behaviors in humans. Little is known of how ASD-risk genes regulate callosal connectivity. Here, we show that Fmr1 , whose loss-of-function leads to Fragile X Syndrome (FXS), cell autonomously promotes maturation of callosal excitatory synapses between somatosensory barrel cortices in mice. Postnatal, cell-autonomous deletion of Fmr1 in postsynaptic Layer (L) 2/3 or L5 neurons results in a selective weakening of AMPA receptor- (R), but not NMDA receptor-, mediated callosal synaptic function, indicative of immature synapses. Sensory deprivation by contralateral whisker trimming normalizes callosal input strength, suggesting that experience-driven activity of postsynaptic Fmr1 KO L2/3 neurons weakens callosal synapses. In contrast to callosal inputs, synapses originating from local L4 and L2/3 circuits are normal, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. These results suggest direct cell autonomous and postnatal roles for FMRP in development of specific cortical circuits and suggest a synaptic basis for long-range functional underconnectivity observed in FXS patients.

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  1. Version published to 10.7554/elife.71555 on eLife
  2. eLife

    Author Response:

    Reviewer #2 (Public Review):

    revious studies showed reduced structural and functional connectivity of two hemispheres in the autism spectrum disorder (ASD), but little is known about its cellular mechanism. This paper tried to fill this knowledge gap using a mouse model of ASD. By combining optogenetics, slice electrophysiology, and Fmr1 gene knockout (KO) approaches (a leading monogenic cause of ASD), the paper demonstrated that callosal inputs to L2/3 pyramidal neurons in whisker somatosensory cortex is reduced in Fmr1 KO mice/neurons compared to wild-type mice/neurons. This reduction was due in part to the selective reduction in AMPA receptor-containing synapses and was restored by sensory input deprivation. The local circuit connection was unaffected in a sparse Fmr1 KO mouse. The paper also recapitulated previously reported the reduced coherence between two hemispheres in vivo. The data is a welcome new addition to the previous studies concerned with circuit abnormality in Fmr1 KO mice and supports the paper's main claim.

    The strength of the paper is the simultaneous comparison of the callosal inputs to L2/3 neurons with or without the Fmr1 gene in the same brain slice. This directly demonstrated the role of the Fmr1 gene on the formation of the callosal synapse, a key piece of information that could guide future basic, translational, and/or therapeutic studies.

    The weakness of the paper is the sparse Fmr1 KO model. An apparent delay in onset of Fmr1 KO effect and unclearness in the extent of Fmr1 KO achieved in one hemisphere makes comparison with global Fmr1 KO model (in this study or previous studies) and assessment of data interpretation made in the paper difficult.

    We include a new supplementary figure (Figure 2 – figure supplement 1) of representative images and quantification of the sparseness of the Cre-GFP expression in our experimental paradigm. We calculate that 3-5% of cells are virally transfected with Cre-GFP. Given the sparseness of Cre-GFP expression, we would expect sparse Fmr1 deletion in other cells in the cortex or other brain regions should be minimal and importantly equally affect inputs onto the recorded postsynaptic Fmr1 KO neurons and their WT neighbors, we think our observation should reliably reflect the function of postsynaptic Fmr1/FMRP.

  3. Version published to 10.1101/2021.06.25.449490v2 on bioRxiv
  4. eLife

    Evaluation Summary:

    The authors find that long-range interhemispheric synapses are selectively weakened following loss of function of the gene mediating fragile X syndrome, the most common inherited form of intellectual disability. Using clever genetic and physiological approaches in mice, the authors show that the effect is cell autonomous and occurs postnatally by impeding the normal developmental strengthening of these synapses. The results convincingly enhance our understanding of the complex pathophysiology of neurological dysfunction in this developmental disorder.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    This is an excellent paper that refines our understanding of how FMRP functions in cortical neurons to shape their synaptic development. The authors use viral channelrhodopsin to selectively activate callosal synapses and find that they are weaker onto L2/3 and L5 pyramidal neurons in the mouse somatosensory cortex. Using sparse deletion, the effect is found to be cell autonomous. Under these conditions it occurs during late development (P23-30) when these synapses are normally being refined and strengthened. The effect is believed to be due to a retention of so-called "silent" synapses which express NMDA receptors but not AMPA receptors, since the input via NMDA receptors is normal and the analysis of evoked quantal events using strontium reveals a reduction in frequency but not a reduction in amplitude. However the existence of silent synapses is not demonstrated directly and the change in frequency observed is small relative to the change in overall callosal synaptic drive. The authors address a prior finding that loss of Fmr1 weakens synapses from L4 neurons onto L2/3 neurons. They find that this does not occur cell autonomously or with postnatal deletion, but does occur in the germline knockout. The authors also find that sensory deprivation via whisker trimming can normalize the callosal input when applied to the whiskers providing the majority of input to the neurons lacking Fmr1, but not when applied to the whiskers providing the majority of input to the contralateral neurons providing the callosal inputs to the KO neurons. This would seem to implicate Fmr1 in an activity-dependent competition between local and long-range synapses. Finally, the authors have reanalyzed prior bilateral EEG recordings in germ-line KO animals and find reduced interhemispheric synchrony, consistent with reduced callosal function. Although these changes could also reflect other effects in the KO brain.

    The experiments are convincing and are well illustrated and analyzed. Taken together they suggest a key role for the regulation of translation by Fmr1 in the activity-dependent refinement of callosal synapses, and likely by extension, many long range connections in the brain. This role could contribute to intellectual disability following loss of Fmr1 function and the sensitivity of this activity-dependent refinement process may explain the frequent observation of callosal abnormalities in other forms of autism spectrum disorders and intellectual disability.

  6. eLife

    Reviewer #2 (Public Review):

    Previous studies showed reduced structural and functional connectivity of two hemispheres in the autism spectrum disorder (ASD), but little is known about its cellular mechanism. This paper tried to fill this knowledge gap using a mouse model of ASD. By combining optogenetics, slice electrophysiology, and Fmr1 gene knockout (KO) approaches (a leading monogenic cause of ASD), the paper demonstrated that callosal inputs to L2/3 pyramidal neurons in whisker somatosensory cortex is reduced in Fmr1 KO mice/neurons compared to wild-type mice/neurons. This reduction was due in part to the selective reduction in AMPA receptor-containing synapses and was restored by sensory input deprivation. The local circuit connection was unaffected in a sparse Fmr1 KO mouse. The paper also recapitulated previously reported the reduced coherence between two hemispheres in vivo. The data is a welcome new addition to the previous studies concerned with circuit abnormality in Fmr1 KO mice and supports the paper's main claim.

    The strength of the paper is the simultaneous comparison of the callosal inputs to L2/3 neurons with or without the Fmr1 gene in the same brain slice. This directly demonstrated the role of the Fmr1 gene on the formation of the callosal synapse, a key piece of information that could guide future basic, translational, and/or therapeutic studies.

    The weakness of the paper is the sparse Fmr1 KO model. An apparent delay in onset of Fmr1 KO effect and unclearness in the extent of Fmr1 KO achieved in one hemisphere makes comparison with global Fmr1 KO model (in this study or previous studies) and assessment of data interpretation made in the paper difficult.

  7. eLife

    Reviewer #3 (Public Review):

    This is an expertly-carried out study that rigorously examines the effects of global and post-synaptic FMR1 deletion on excitatory synaptic function in primary sensory cortex. These data add to a growing literature about the effects of FMR1 on different subnetworks in the brain, including that from the authors' own labs. Results indicate that L5 neurons show hyperconnectivity within the layer in the global KO, but the postsynaptic effects of FMR1 deletion are only transiently manifested at the L4 to L2/3 connections, and local connections between L2/3 neurons are not affected. Interestingly, callosal inputs show selective impairment into the 4th postnatal week, and mosaic knock-out of FMR1 indicates that this effect is postsynaptic in origin. Although the degree of specificity by which this gene can influence different types of excitatory synapses is provocative (i.e. short-range inputs appear mostly normal in superficial layers but long-range callosal inputs are depressed), the mechanism for this selectivity remains obscure. Although the authors suggest that this reduction in synaptic strength can drive reduced coherence in the interhemispheric EEG, important controls for the analysis of EEG coherence and the circuit relationship between callosal inputs and the EEG are missing, such as the effect of FMR1 KO on inhibitory neurons, as well as lack of correspondence in age- and brain region between the synaptic and EEG studies.

  8. Version published to 10.1101/2021.06.25.449490v1 on bioRxiv