FOXP1 negatively regulates intrinsic excitability in D2 striatal projection neurons by promoting inwardly rectifying and leak potassium currents.

Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.

Local cortical circuit correlates of altered EEG in the mouse model of Fragile X syndrome.

Electroencephalogram (EEG) recordings in Fragile X syndrome (FXS) patients have revealed enhanced sensory responses, enhanced resting "gamma frequency" (30-100 Hz) activity, and a decreased ability for sensory stimuli to modulate cortical activity at gamma frequencies. Similar changes are observed in the FXS model mouse - the Fmr1 knockout. These alterations may become effective biomarkers for diagnosis and treatment of FXS. Therefore, it is critical to better understand what circuit properties underlie these changes. We employed Channelrhodopsin2 to optically activate local circuits in the auditory cortical region in brain slices to examine how changes in local circuit function may be related to EEG changes. We focused on layers 2/3 and 5 (L2/3 and L5). In Fmr1 knockout mice, light-driven excitation of L2/3 revealed hyperexcitability and increased gamma frequency power in both local L2/3 and L5 circuits. Moreover, there is increased synchrony in the gamma frequency band between L2/3 and L5. Hyperexcitability and increased gamma power were not observed in L5 with L5 light-driven excitation, indicating that these changes were layer-specific. A component of L2/3 network hyperexcitability is independent of ionotropic receptor mediated synaptic transmission and may be mediated by increased intrinsic excitability of L2/3 neurons. Finally, lovastatin, a candidate therapeutic compound for FXS that targets ERK signaling did not normalize changes in gamma activity. In conclusion, hyperactivity and increased gamma activity in local neocortical circuits, together with increased gamma synchrony between circuits, provide a putative substrate for EEG alterations observed in both FXS patients and the FXS mouse model.

Palmitoylation and Membrane Binding of Arc/Arg3.1: A Potential Role in Synaptic Depression.

Activity-regulated cytoskeletal-associated protein (Arc, also known as activity-regulated gene 3.1 or Arg3.1) is induced in neurons in response to salient experience and neural activity and is necessary for activity-induced forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), cellular substrates of learning and memory. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors in dendritic spines, a process associated with LTD. Arc has also been implicated in the proteolytic processing of amyloid precursor protein on the surface of endosomes. To mediate these activities, Arc must associate with cellular membranes, but it is unclear whether Arc binds directly to the lipid bilayer or requires protein-protein interactions for membrane recruitment. In this study, we show that Arc associates with pure phospholipid vesicles in vitro and undergoes palmitoylation in neurons, a modification that allows it to insert directly into the hydrophobic core of the bilayer. The palmitoylated cysteines are clustered in a motif, 94CLCRC98, located in the N-terminal half of the protein, which has not yet been structurally characterized. Expression of Arc with three mutated cysteines in that motif cannot support synaptic depression induced by the activity-dependent transcription factor, MEF2 (myocyte enhancer factor 2), in contrast to wild-type Arc. Thus, it appears that palmitoylation regulates at least a subset of Arc functions in synaptic plasticity.

Vagus Nerve Stimulation Enhances Extinction of Conditioned Fear in Rats and Modulates Arc Protein, CaMKII, and GluN2B-Containing NMDA Receptors in the Basolateral Amygdala.

Vagus nerve stimulation (VNS) enhances the consolidation of extinction of conditioned fear. High frequency stimulation of the infralimbic cortex (IL) produces long-term potentiation in the basolateral amygdala (BLA) in rats given VNS-paired extinction training, whereas the same stimulation produces long-term depression in sham-treated rats. The present study investigated the state of synaptic plasticity-associated proteins in the BLA that could be responsible for this shift. Male Sprague-Dawley rats were separated into 4 groups: auditory fear conditioning only (fear-conditioned); fear conditioning + 20 extinction trials (extended-extinction); fear conditioning + 4 extinction trials paired with sham stimulation (sham-extinction); fear conditioning + 4 extinction trials paired with VNS (VNS-extinction). Freezing was significantly reduced in extended-extinction and VNS-extinction rats. Western blots were used to quantify expression and phosphorylation state of synaptic plasticity-associated proteins such as Arc, CaMKII, ERK, PKA, and AMPA and NMDA receptors. Results show significant increases in GluN2B expression and phosphorylated CaMKII in BLA samples from VNS- and extended-extinction rats. Arc expression was significantly reduced in VNS-extinction rats compared to all groups. Administration of the GluN2B antagonist ifenprodil immediately after fear extinction training blocked consolidation of extinction learning. Results indicate a role for BLA CaMKII-induced GluN2B expression and reduced Arc protein in VNS-enhanced extinction.