Altered integration of excitatory inputs onto the basal dendrites of layer 5 pyramidal neurons in a mouse model of Fragile X syndrome

Abstract

Layer 5 (L5) pyramidal neurons receive predictive and sensory inputs in a compartmentalized manner at their apical and basal dendrites, respectively. To uncover how integration of sensory inputs is affected in autism spectrum disorders (ASD), we used two-photon glutamate uncaging to activate spines in the basal dendrites of L5 pyramidal neurons from a mouse model of Fragile X syndrome (FXS), the most common genetic cause of ASD. While subthreshold excitatory inputs integrate linearly in wild-type animals, surprisingly those with FXS summate sublinearly, contradicting what would be expected of sensory hypersensitivity classically associated with ASD. We next investigated the mechanism underlying this sublinearity by performing knockdown of the regulatory β4 subunit of BK channels, which rescued the synaptic integration, a result that was corroborated with numerical simulations. Taken together, these findings suggest that there is a differential impairment in the integration of feedforward sensory and feedback predictive inputs in L5 pyramidal neurons in FXS and potentially other forms of ASD, as a result of specifically localized subcellular channelopathies. These results challenge the traditional view that FXS and other ASD are characterized by sensory hypersensitivity, proposing instead a hyposensitivity of sensory inputs and hypersensitivity of predictive inputs onto cortical neurons.

Keywords: BK channels; autism spectrum disorders; dendritic spines; layer 5 pyramidal neurons; synaptic integration.

Fig. 1.

Sublinear summation of excitatory synaptic inputs in basal dendrites of Fmr1-KO L5 pyramidal neurons. (A) Representative basal dendrite selected for 2P glutamate uncaging to activate spines from WT (Top) and Fmr1-KO L5 pyramidal neurons (Bottom). Green dots indicate the site for uncaging. Spines were first activated individually (Spine 1 or Spine 2) and then together (Spine 1 & Spine 2). Blue dashed traces correspond to the linear sum of individual events of each spine. (B) Observed response has smaller amplitude, integral and gain than expected based on the sum of individual responses in Fmr1-KO mice, while in WT mice, the responses summate linearly. (C) As in B, but values were averaged per mouse.

Fig. 2.

Expression of αBK and β4 subunits in synapses of Fmr1-KO mice. (A and B) Mouse visual cortex TP and Synaptoneurosome (SN) fractions were analyzed by western blot with specific monoclonal antibodies and normalized by the expression of γ-tubulin. (A) Expression of αBK (20 µg/line). Synaptic enrichment in the SN fraction was confirmed by the expression of the postsynaptic density (PSD) marker PSD95. (B) αBK expression normalized by γ-tubulin and by the expression in WT/TP (n = 3, P < 0.0001, ANOVA). (C) Expression of β4. A mix of three samples for each condition was loaded at decreasing concentrations (24, 12, 6 µg/line). (D) β4 expression normalized by γ-tubulin and by the expression in WT/TP (n = 3, P < 0.05, P < 0.0001, ANOVA). (E) Representation of the experimental workflow (see Methods) and blots corresponding to co-IP of FMRP, immunoprecipitation (IP) of β4 and the loading control γ-tubulin (200 μg/sample input protein). Left side of the bottom panel, co-IP Western blot performed using anti-β4 antibody to precipitate and detect a protein complex containing FMRP bound to β4 subunit in the visual cortex and hippocampus of Fmr1-KO and control mice P25±1 (n = 3, pooled samples). Right side (pre-IP) shows the composition of the original sample (16 μg/line). (F–I) Expression of channel subunits in excitatory synapses was studied in P26±1 Fmr1-KO and control transgenic mice expressing GFP in L5 pyramidal neuron (green) by immunofluorescent detection of αBK (F) and β4 subunits (red) (G) Dendritic spine channel expression is shown in one <1-μm confocal optical slice. Arrow heads indicate spines with detectable expression of the BK subunit. Insets (yellow boxes) and additional examples at higher magnification are shown below each group with clear αBK or β4 subunit spine localization. (Scale bar, 1 μm.) (H) Examples of dendrite 3D reconstructions from the same neurons shown in G), above threshold β4 subunit immunoreactivity and signal overlapping with the spine head are shown as red dots. (I) Mean intensity of the β4 subunit signal in each spine head was compared between Fmr1-KO and control mice. (Fmr1-KO, n = 206 spines, WT, n = 143 spines, P < 0.05, Mann–Whitney test).

Fig. 3.

Knock-down of β4 subunit of BK channels rescues sublinearity of synaptic inputs in the basal dendrites of Fmr1-KO L5 pyramidal neurons. (A) Schematic of the shRNA vector used to knockdown β4. (B and C) shRNA knock-down of the β4 subunit of BK channels rescues the sublinearity in the basal dendrites of Fmr1-KO L5 pyramidal neurons (B) while injection of scrambled shRNA has no effect (C). (D) Observed responses were not significantly different in amplitude, integral, and gain than that expected based on the sum of individual responses in Fmr1-KO mice injected with the β4shRNA, while those injected with a scrambled version, the responses summate sublinearly. (E) As in D, but values were averaged per mouse. (F and G) Spine density (F) and spine morphology (G) are not significantly different in Fmr1-KO L5 pyramidal neurons injected with shRNA targeted to the β4 subunit of BK channels versus nonspecific scrambled shRNA in L5 of the visual cortex.

Fig. 4.

Biophysical modeling shows the effect of the BK channel β4 subunit on synaptic integration in dendritic spines. (A and B) Left panels: Schematic of the model used to assess synaptic integration in the basal dendrites of a WT L5 pyramidal neuron (A) expressing FMRP, which sequesters the β4 subunit so only the α subunit is present in the spine and a Fmr1-KO L5 pyramidal neuron (B), which does not express FMRP so the β4 subunit is free to bind the BK channel. Right panels: Spines were first activated individually (Spine 1 or Spine 2) and then together (Spine 1 & Spine 2), and the EPSP generated at the soma was recorded. Blue dashed traces correspond to the linear sum of individual events of each spine. In the WT neuron, the actual and expected EPSP responses match (compare black and dashed blue traces), whereas in the KO neuron, the actual EPSP response is much less than expected (compare dashed red and blue traces). (C) Observed response has smaller amplitude, integral, and gain than the expected based on the sum of individual responses in Fmr1-KO neuron, while in WT mice, the responses summate linearly. (D) Number of BK channels open during activation of 1 spine or 2 spines in the modeled WT and Fmr1-KO L5 pyramidal neuron.