Autism spectrum disorder-like behavior caused by reduced excitatory synaptic transmission in pyramidal neurons of mouse prefrontal cortex

Abstract

Autism spectrum disorder (ASD) is thought to result from deviation from normal development of neural circuits and synaptic function. Many genes with mutation in ASD patients have been identified. Here we report that two molecules associated with ASD susceptibility, contactin associated protein-like 2 (CNTNAP2) and Abelson helper integration site-1 (AHI1), are required for synaptic function and ASD-related behavior in mice. Knockdown of CNTNAP2 or AHI1 in layer 2/3 pyramidal neurons of the developing mouse prefrontal cortex (PFC) reduced excitatory synaptic transmission, impaired social interaction and induced mild vocalization abnormality. Although the causes of reduced excitatory transmission were different, pharmacological enhancement of AMPA receptor function effectively restored impaired social behavior in both CNTNAP2- and AHI1-knockdown mice. We conclude that reduced excitatory synaptic transmission in layer 2/3 pyramidal neurons of the PFC leads to impaired social interaction and mild vocalization abnormality in mice.

Combining electrophysiology and optogenetics for functional screening of pyramidal neurons in the mouse prefrontal cortex

“…The neurons expressing GFP or RFP can be judged to be layer 2/3 pyramidal neurons because the layer showing the fluorescence is determined according to the date of the embryonic stages in which the in utero electroporation was performed ( Fukuchi-Shimogori and Grove, 2001 ; Saito, 2006 ). Note: As far as we know, around 30% of the electroporated neurons are CaMKII-positive neurons in the mPFC ( Nagahama et al., 2020 ; Sacai et al., 2020 ). Put the patch pipette on the targeted neuron with positive pressure during approaching the neuron.…”

“… Note: If any abnormalities in either amplitude or frequency are found, we recommend recording the evoked synaptic responses by local electrical stimulation using a bipolar tungsten stimulating electrode as the next step. The magnitude of the amplitude and paired-pulse synaptic responses should be recorded ( Nagahama et al., 2020 ; Sacai et al., 2020 ) ( troubleshooting 3 ). Note: When applying the electrical stimulation, the bipolar tungsten stimulating electrode should be placed 50–100 μm away from recorded neurons to prevent the direct stimulation of neurons ( Nagahama et al., 2020 ; Sacai et al., 2020 ).…”

“…The magnitude of the amplitude and paired-pulse synaptic responses should be recorded ( Nagahama et al., 2020 ; Sacai et al., 2020 ) ( troubleshooting 3 ). Note: When applying the electrical stimulation, the bipolar tungsten stimulating electrode should be placed 50–100 μm away from recorded neurons to prevent the direct stimulation of neurons ( Nagahama et al., 2020 ; Sacai et al., 2020 ). CRITICAL: Keep the same distances between the electrode and a pair of the neurons with and without fluorescent proteins especially when analyzing the amplitude of the evoked synaptic responses because this directly affects the magnitude of the evoked synaptic responses.…”

“… CRITICAL: Keep the same distances between the electrode and a pair of the neurons with and without fluorescent proteins especially when analyzing the amplitude of the evoked synaptic responses because this directly affects the magnitude of the evoked synaptic responses. Note: When recording the paired-pulse synaptic responses induced by the electrical stimulation, it might be better to record them at several inter-stimulus intervals such as 20, 50, 100, and 500 msec ( Nagahama et al., 2020 ; Sacai et al., 2020 ). If any impairments in the initial paired-pulse ratio (PPR) are found, record the paired-pulse responses at 20, 50, 100, 500 ms inter-stimulus intervals (ISI) by the 0.1-ms electrical stimulation and 2 to 5-ms light shining to identify which pre or postsynaptic site is affected by the KD of targeted gene ( Nagahama et al., 2020 ).…”

“…For complete details on the use and execution of this protocol, please refer to Nagahama et al. (2020) and Sacai et al. (2020) .…”