The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking.

Neuroimmune interactions play a significant role in regulating synaptic plasticity in both the healthy and diseased brain. The complement pathway, an extracellular proteolytic cascade, exemplifies these interactions. Its activation triggers microglia-dependent synaptic elimination via the complement receptor 3 (CR3). Current models of pathological complement activity in the brain propose that accelerated synaptic loss resulting from overexpression of C4 (C4-OE), a gene associated with schizophrenia, follows this pathway. Here, we report that C4-mediated cortical hypoconnectivity is CR3-independent. Instead, C4-OE triggers impaired GluR1 trafficking through an intracellular mechanism involving the endosomal protein SNX27, resulting in pathological synaptic loss. Moreover, C4 circuit alterations in the prefrontal cortex, a brain region associated with neuropsychiatric disorders, were rescued by increasing neuronal levels of SNX27, which we identify as an interacting partner of this neuroimmune protein. Our results link excessive complement activity to an intracellular endo-lysosomal trafficking pathway altering synaptic plasticity.

REVEALS: an open-source multi-camera GUI for rodent behavior acquisition.

Deciphering the rich repertoire of mouse behavior is crucial for understanding the functions of both the healthy and diseased brain. However, the current landscape lacks effective, affordable, and accessible methods for acquiring such data, especially when employing multiple cameras simultaneously. We have developed REVEALS (Rodent Behavior Multi-Camera Laboratory Acquisition), a graphical user interface for acquiring rodent behavioral data via commonly used USB3 cameras. REVEALS allows for user-friendly control of recording from one or multiple cameras simultaneously while streamlining the data acquisition process, enabling researchers to collect and analyze large datasets efficiently. We release this software package as a stand-alone, open-source framework for researchers to use and modify according to their needs. We describe the details of the graphical user interface implementation, including the camera control software and the video recording functionality. We validate results demonstrating the graphical user interface's stability, reliability, and accuracy for capturing rodent behavior using DeepLabCut in various behavioral tasks. REVEALS can be incorporated into existing DeepLabCut, MoSeq, or other custom pipelines to analyze complex behavior. In summary, REVEALS offers an interface for collecting behavioral data from single or multiple perspectives, which, when combined with deep learning algorithms, enables the scientific community to identify and characterize complex behavioral phenotypes.

Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex.

A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.

Schizophrenia is a severe mental disorder with an unclear pathophysiology. Increased expression of the immune gene C4 has been linked to a greater risk of developing schizophrenia; however, it is not known whether C4 plays a causative role in this brain disorder. Using confocal imaging and whole-cell electrophysiology, we demonstrate that overexpression of C4 in mouse prefrontal cortex neurons leads to perturbations in dendritic spine development and hypoconnectivity, which mirror neuropathologies found in schizophrenia patients. We find evidence that microglia-mediated synaptic engulfment is enhanced with increased expression of C4. We also show that C4-dependent circuit dysfunction in the frontal cortex leads to decreased social interactions in juvenile and adult mice. These results demonstrate that increased expression of the schizophrenia-associated gene C4 causes aberrant circuit wiring in the developing prefrontal cortex and leads to deficits in juvenile and adult social behavior, suggesting that altered C4 expression contributes directly to schizophrenia pathogenesis.

Overexpression of the schizophrenia risk gene C4 in PV cells drives sex-dependent behavioral deficits and circuit dysfunction.

Fast-spiking parvalbumin (PV)-positive cells are key players in orchestrating pyramidal neuron activity, and their dysfunction is consistently observed in myriad brain diseases. To understand how immune complement dysregulation - a prevalent locus of brain disease etiology - in PV cells may drive disease pathogenesis, we have developed a transgenic mouse line that permits cell-type specific overexpression of the schizophrenia-associated complement component 4 (C4) gene. We found that overexpression of mouse C4 (mC4) in PV cells causes sex-specific behavioral alterations and concomitant deficits in synaptic connectivity and excitability of PV cells of the prefrontal cortex. Using a computational network, we demonstrated that these microcircuit deficits led to hyperactivity and disrupted neural communication. Finally, pan-neuronal overexpression of mC4 failed to evoke the same deficits in behavior as PV-specific mC4 overexpression, suggesting that C4 perturbations in fast-spiking neurons are more harmful to brain function than pan-neuronal alterations. Together, these results provide a causative link between C4 and the vulnerability of PV cells in brain disease.

Overexpression of the schizophrenia risk gene C4 in PV cells drives sex-dependent behavioral deficits and circuit dysfunction.

Fast-spiking parvalbumin (PV)-positive cells are key players in orchestrating pyramidal neuron activity, and their dysfunction is consistently observed in myriad brain diseases. To understand how immune complement pathway dysregulation in PV cells drives disease pathogenesis, we have developed a transgenic line that permits cell-type specific overexpression of the schizophrenia-associated C4 gene. We found that overexpression of mouse C4 (mC4) in PV cells causes sex-specific alterations in anxiety-like behavior and deficits in synaptic connectivity and excitability of PFC PV cells. Using a computational model, we demonstrated that these microcircuit deficits led to hyperactivity and disrupted neural communication. Finally, pan-neuronal overexpression of mC4 failed to evoke the same deficits in behavior as PV-specific mC4 overexpression, suggesting that perturbations of this neuroimmune gene in fast-spiking neurons are especially detrimental to circuits associated with anxiety-like behavior. Together, these results provide a causative link between C4 and the vulnerability of PV cells in brain disease.

A pipeline for STED super-resolution imaging and Imaris analysis of nanoscale synapse organization in mouse cortical brain slices.

Advances in super-resolution imaging enable us to delve into its intricate structural and functional complexities with unprecedented detail. Here, we present a pipeline to visualize and analyze the nanoscale organization of cortical layer 1 apical dendritic spines in the mouse prefrontal cortex. We describe steps for brain slice preparation, immunostaining, stimulated emission depletion super-resolution microscopy, and data analysis using the Imaris software package. This protocol allows the study of physiologically relevant brain circuits implicated in neuropsychiatric disorders.

REVEALS: An Open Source Multi Camera GUI For Rodent Behavior Acquisition.

Understanding the rich behavioral data generated by mice is essential for deciphering the function of the healthy and diseased brain. However, the current landscape lacks effective, affordable, and accessible methods for acquiring such data, especially when employing multiple cameras simultaneously. We have developed REVEALS (Rodent BEhaVior Multi-camErA Laboratory AcquiSition), a graphical user interface (GUI) written in python for acquiring rodent behavioral data via commonly used USB3 cameras. REVEALS allows for user-friendly control of recording from one or multiple cameras simultaneously while streamlining the data acquisition process, enabling researchers to collect and analyze large datasets efficiently. We release this software package as a stand-alone, open-source framework for researchers to use and modify according to their needs. We describe the details of the GUI implementation, including the camera control software and the video recording functionality. We validate results demonstrating the GUI's stability, reliability, and accuracy for capturing and analyzing rodent behavior using DeepLabCut pose estimation in both an object and social interaction assay. REVEALS can also be incorporated into other custom pipelines to analyze complex behavior, such as MoSeq. In summary, REVEALS provides an interface for collecting behavioral data from one or multiple perspectives that, combined with deep learning algorithms, will allow the scientific community to discover and characterize complex behavioral phenotypes to understand brain function better.

C4 induces pathological synaptic loss by impairing AMPAR trafficking.

During development, activation of the complement pathway, an extracellular proteolytic cascade, results in microglia-dependent synaptic elimination via complement receptor 3 (CR3). Here, we report that decreased connectivity caused by overexpression of C4 (C4-OE), a schizophrenia-associated gene, is CR3 independent. Instead, C4-OE triggers GluR1 degradation through an intracellular mechanism involving endosomal trafficking protein SNX27, resulting in pathological synaptic loss. Moreover, the connectivity deficits associated with C4-OE were rescued by increasing levels of SNX27, linking excessive complement activity to an intracellular endolysosomal recycling pathway affecting synapses.

Sex Differences in Behavioral and Brainstem Transcriptomic Neuroadaptations following Neonatal Opioid Exposure in Outbred Mice.

The opioid epidemic led to an increase in the number of neonatal opioid withdrawal syndrome (NOWS) cases in infants born to opioid-dependent mothers. Hallmark features of NOWS include weight loss, severe irritability, respiratory problems, and sleep fragmentation. Mouse models provide an opportunity to identify brain mechanisms that contribute to NOWS. Neonatal outbred Swiss Webster Cartworth Farms White (CFW) mice were administered morphine (15 mg/kg, s.c.) twice daily from postnatal day 1 (P1) to P14, an approximation of the third trimester of human gestation. Female and male mice underwent behavioral testing on P7 and P14 to determine the impact of opioid exposure on anxiety and pain sensitivity. Ultrasonic vocalizations (USVs) and daily body weights were also recorded. Brainstems containing pons and medulla were collected during morphine withdrawal on P14 for RNA sequencing. Morphine induced weight loss from P2 to P14, which persisted during adolescence (P21) and adulthood (P50). USVs markedly increased at P7 in females, emerging earlier than males. On P7 and P14, both morphine-exposed female and male mice displayed hyperalgesia on the hot plate and tail-flick assays, with females showing greater hyperalgesia than males. Morphine-exposed mice exhibited increased anxiety-like behavior in the open-field arena on P21. Transcriptome analysis of the brainstem, an area implicated in opioid withdrawal and NOWS, identified pathways enriched for noradrenergic signaling in females and males. We also found sex-specific pathways related to mitochondrial function and neurodevelopment in females and circadian entrainment in males. Sex-specific transcriptomic neuroadaptations implicate unique neurobiological mechanisms underlying NOWS-like behaviors.