GABAergic circuits are critical for the synchronization and higher order function of brain networks. Defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons where it interacts directly with the GABA A receptor associated protein (GABARAP) to stabilize cell surface GABA A receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction ( Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-of-function variants may contribute to human disease.

Summary Long-lasting forms of synaptic plasticity such as synaptic scaling are critically dependent on transcription. Activity-dependent transcriptional dynamics in neurons, however, remain incompletely characterized because most previous efforts relied on measurement of steady-state mRNAs. Here, we use nascent RNA sequencing to profile transcriptional dynamics of primary neuron cultures undergoing network activity shifts. We find pervasive transcriptional changes, in which ∼45% of expressed genes respond to network activity shifts. We further link retinoic acid-induced 1 (RAI1), the Smith-Magenis syndrome gene, to the transcriptional program driven by reduced network activity. Remarkable agreement among nascent transcriptomes, dynamic chromatin occupancy of RAI1, and electrophysiological properties of Rai1 -deficient neurons demonstrates the essential roles of RAI1 in suppressing synaptic upscaling in the naive network, while promoting upscaling triggered by activity silencing. These results highlight the utility of bona fide transcription profiling to discover mechanisms of activity-dependent chromatin remodeling that underlie normal and pathological synaptic plasticity.

GABAergic circuits are critical for the synchronization and higher order function of brain networks, and defects in this circuitry are linked to neuropsychiatric diseases, including bipolar disorder, schizophrenia, and autism. Work in cultured neurons has shown that ankyrin-G plays a key role in the regulation of GABAergic synapses on the axon initial segment and somatodendritic domain of pyramidal neurons where it interacts directly with the GABAA receptor associated protein (GABARAP) to stabilize cell surface GABAA receptors. Here, we generated a knock-in mouse model expressing a mutation that abolishes the ankyrin-G/GABARAP interaction (Ank3 W1989R) to understand how ankyrin-G and GABARAP regulate GABAergic circuitry in vivo. We found that Ank3 W1989R mice exhibit a striking reduction in forebrain GABAergic synapses resulting in pyramidal cell hyperexcitability and disruptions in network synchronization. In addition, we identified changes in pyramidal cell dendritic spines and axon initial segments consistent with compensation for hyperexcitability. Finally, we identified the ANK3 W1989R variant in a family with bipolar disorder, suggesting a potential role of this variant in disease. Our results highlight the importance of ankyrin-G in regulating forebrain circuitry and provide novel insights into how ANK3 loss-offunction variants may contribute to human disease. molecular mechanisms underlying the subcellular organization of cortical GABAergic synapses remain poorly understood. Abnormalities in GABAergic interneuron circuitry and decreased gamma oscillations have been implicated in many neurodevelopmental and neuropsychiatric disorders 1-8 . Thus, the understanding of the cellular and molecular mechanisms that contribute to the development and function of GABAergic synapses as well as identification of genetic variants that contribute to neuropsychiatric disorders is critical to the discovery of new therapeutic agents for the treatment of diseases involving altered inhibitory circuits.ANK3 encodes ankyrin-G, a fundamental scaffolding protein that organizes critical plasma membrane domains 9, 10 . Alternative splicing of ANK3 in the brain gives rise to three main isoforms of ankyrin-G: the canonical 190 kDa isoform, a 270 kDa isoform, and a giant, 480 kDa isoform. The 190 kDa isoform is expressed in most tissues and cell types throughout the body including brain, heart, skeletal muscle, kidney, and retina. The 270 kDa and 480 kDa isoforms of ankyrin-G are predominantly expressed in the nervous system, and arise from alternative splicing of a 7.8 kb vertebrate-specific exon 9, 11 . The 480 kDa ankyrin-G isoform has been identified as the master organizer of axon initial segments (AIS) and nodes of Ranvier, sites of action potential (AP) initiation and propagation 10 . This splice variant is necessary for the proper clustering of voltage-gated sodium channels, KCNQ2/3 potassium channels, the cell adhesion molecule neurofascin-186, and the cytoskeletal protein βIV-spectrin to excitable domains (reviewed in 12 )....

Gamma-band oscillations (GBOs) are generated by fast-spiking interneurons and are critical for cognitive functions. Abnormalities in GBOs are frequently observed in schizophrenia and bipolar disorder and are strongly correlated with cognitive impairment. However, the underlying mechanisms are poorly understood. Studying GBOs inex vivopreparations is challenging due to high energy demands and the need for continuous oxygen delivery to the tissue. As a result, GBOs are typically studied in brain tissue from very young animals or in experimental setups that maximize oxygen supply but compromise spatial resolution. Thus, there is a limited understanding of how GBOs interact within and between different brain structures remains or in brain tissue from mature animals. To address these limitations, we have developed a novel approach for studying GBOs inex vivohippocampal slices from mature animals, utilizing 60-channel, perforated microelectrode arrays (pMEAs). pMEAs enhance oxygen delivery and increase spatial resolution in electrophysiological recordings, enabling comprehensive analyses of GBO synchronization within discrete brain structures. We found that transecting the Schaffer collaterals, a neural pathway within the hippocampus, impairs GBO coherence between CA1 and CA3 subfields. Furthermore, we validated our approach by studying GBO coherence in an Ank3 mutant mouse model exhibiting inhibitory synaptic dysfunction. We discovered that GBO coherence remains intact in the CA3 subfield of these mutant mice but is impaired within and between the CA1 subfield. Overall, our approach offers significant potential to characterize GBOs inex vivobrain sections of animal models, enhancing our understanding of network dysfunction in psychiatric disorders.Significance StatementSynchronized brain activity is crucial for various cognitive behaviors, and abnormalities in gamma-band oscillations (GBOs) are prevalent in numerous mental health disorders. Our study presents an innovative method that utilizes microelectrode arrays to record GBOs across multiple locations within the hippocampus. This approach allows us to investigate the development of GBO coherence within and between specific subregions of the hippocampus, providing a more comprehensive understanding of how brain activity is synchronized in both healthy rodents and animal models of neurological and psychiatric diseases.

Oscillations play crucial roles in many cognitive processes such as memory formation and attention. GABAergic interneurons can synchronize neuronal activity leading to gamma oscillations (30-60 Hz). Abnormalities in oscillatory activity in the hippocampus have been implicated in the pathology of some mental health disorders including schizophrenia and bipolar disorder, however the neurobiological mechanism underlying these abnormal oscillations are not yet fully understood. We set out to develop a reliable approach to study gamma oscillations in ex vivo hippocampal preparations using perforated microelectrode arrays. Perforated microelectrode arrays allow for the simultaneous measurement of electrical activity at multiple sites while allowing solutions to pass through the brain section. We obtained extracellular electrophysiological recordings from acute sections of mouse hippocampus situated on a 60-channel, perforated microelectrode arrays (pMEAs). Bath application of kainate rapidly induced and maintained oscillatory activity in the CA1 and CA3 regions of the hippocampus. Kainate-induced oscillations were quickly abolished by the GABAA receptor antagonist, bicuculline. Furthermore, we employed this approach on a mouse model of bipolar disorder. Sections prepared from mutant mice exhibited an increase in the coherence of gamma power within CA1 despite a reduction in gamma band power.