Rhushikesh A. Phadke

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.

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.

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.

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.