We demonstrate editing of post-mitotic neurons in the adult mouse brain following injection of Cas9 ribonucleoprotein (RNP) complexes in the hippocampus, striatum and cortex. Engineered variants of Cas9 with multiple SV40 nuclear localization sequences enabled a tenfold increase in the efficiency of neuronal editing in vivo. These advances indicate the potential of genome editing in the brain to correct or inactivate the underlying genetic causes of neurological diseases.

Stress regulation of brain-derived neurotrophic factor (BDNF) is implicated in the hippocampal damage observed in depression. BDNF has a complex gene structure with four 5 0 untranslated exons (I-IV) with unique promoters, and a common 3 0 coding exon (V). To better understand the stress regulation of BDNF, we addressed whether distinct stressors differentially regulate exon-specific BDNF transcripts in the postnatal and adult hippocampus. The early life stress of maternal separation (MS) resulted in a time point-dependent differential upregulation of BDNF transcripts restricted to early postnatal life (P14-BDNF II, P21-BDNF IV, V). In adulthood, distinct stressors regulated BDNF transcripts in a signature manner. Immobilization stress, administered once, decreased all BDNF splice variants but had differing effects on BDNF I/II (increase) and III/IV (decrease) when administered chronically. Although immobilization stress reduced BDNF (V) mRNA, chronic unpredictable stress did not influence total BDNF despite altering specific BDNF transcripts. Furthermore, a prior history of MS altered the signature pattern in which adult-onset stress regulated specific BDNF transcripts. We also examined the expression of cyclic AMP response element-binding protein (CREB), an upstream transcriptional activator of BDNF, and observed a CREB induction in the postnatal hippocampus following MS. As a possible consequence of enhanced CREB and BDNF expression following MS, we examined hippocampal progenitor proliferation and observed a significant increase restricted to early life. These results suggest that alterations in CREB/BDNF may contribute to the generation of individual differences in stress neurocircuitry, providing a substrate for altered vulnerability to depressive disorders.

Prefrontal serotonin 5-HT 2 receptors have been linked to the pathogenesis and treatment of affective disorders, yet their function in psychiatric vulnerability is not known. Here, we examine the effects of 5-HT 2 receptors in a rat model of psychiatric vulnerability using electrophysiology, gene expression, and behavior. Following the early stress of chronic maternal separation, we found that serotonin has atypical 5-HT 2 receptor-mediated excitatory effects in the adult prefrontal cortex that were blocked by the 5-HT 2A receptor antagonist MDL 100907. In the absence of a serotonergic agonist, the intrinsic excitability of the prefrontal cortex was not enhanced relative to controls. Yet, in response to stimulation of 5-HT 2 receptors, adult animals with a history of early stress exhibit heightened prefrontal network activity in vitro, enhanced immediate early gene expression in vivo, and potentiated head shake behavior. These changes arise in the absence of any major alteration of prefrontal 5-HT 2A/C mRNA expression or 5-HT 2 receptor binding. Our microarray results and quantitative PCR validation provide insight into the molecular changes that accompany such enhanced 5-HT 2 receptor function in adult animals following early stress. We observed persistent prefrontal transcriptome changes, with significant enrichment of genes involved in cellular developmental processes, regulation of signal transduction, and G-protein signaling. Specific genes regulated by early stress were validated in an independent cohort, and several altered genes were normalized by chronic blockade of 5-HT 2 receptors in adulthood. Together, our results demonstrate enhanced prefrontal 5-HT 2 receptor function and persistent alterations in prefrontal gene expression in a rat model of psychiatric vulnerability.

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