The altered sensitivity of acute stress induced anxiety-related behaviors by modulating insular cortex-paraventricular thalamus-bed nucleus of the stria terminalis neural circuit.

The dysregulation of neuronal networks contributes to the etiology of psychiatric diseases, including anxiety. However, the neural circuits underlying anxiety symptoms remain unidentified. We observed acute restraint stress activating excitatory neurons in the paraventricular thalamus (PVT). Activation of PVT neurons caused anxious behaviors, whereas suppression of PVT neuronal activity induced an anxiolytic effect, achieved by using a chemogenetic method. Moreover, we found that the PVT neurons showed plentiful neuronal projections to the bed nucleus of the stria terminalis (BNST). Activation of PVT-BNST neural projections increased the susceptibility of stress-induced anxiety-related behaviors, and inhibition of this neural circuit produced anxiolysis. The insular cortex (IC) is an important upstream region projecting to PVT. Activation of IC-PVT neuronal projections enhanced susceptibility to stress induced anxious behaviors. Inhibiting this neural circuit suppressed anxious behaviors. Moreover, anterograde monosynaptic tracing results showed that the IC exerts strong neuronal projections to PVT, forming synaptic connections with its neurons, and these neurons throw extensive neuronal fibers to form synapse with BNST neurons. Finally, our results showed that ablation of neurons in PVT receiving monosynaptic input from IC attenuated the anxiety-related phenotypes induced by activating IC neurons. Lesions of the neurons in BNST synaptic origination from PVT blocked the anxiety-related phenotypes induced by activating PVT neurons. Our findings indicate that the PVT is a crucial anxiety-regulating nucleus, and the IC-PVT-BNST neural projection is an essential pathway affecting anxiety morbidity and treatment.

Deciphering an AgRP-serotoninergic neural circuit in distinct control of energy metabolism from feeding.

Contrasting to the established role of the hypothalamic agouti-related protein (AgRP) neurons in feeding regulation, the neural circuit and signaling mechanisms by which they control energy expenditure remains unclear. Here, we report that energy expenditure is regulated by a subgroup of AgRP neurons that send non-collateral projections to neurons within the dorsal lateral part of dorsal raphe nucleus (dlDRN) expressing the melanocortin 4 receptor (MC4R), which in turn innervate nearby serotonergic (5-HT) neurons. Genetic manipulations reveal a bi-directional control of energy expenditure by this circuit without affecting food intake. Fiber photometry and electrophysiological results indicate that the thermo-sensing MC4RdlDRN neurons integrate pre-synaptic AgRP signaling, thereby modulating the post-synaptic serotonergic pathway. Specifically, the MC4RdlDRN signaling elicits profound, bi-directional, regulation of body weight mainly through sympathetic outflow that reprograms mitochondrial bioenergetics within brown and beige fat while feeding remains intact. Together, we suggest that this AgRP neural circuit plays a unique role in persistent control of energy expenditure and body weight, hinting next-generation therapeutic approaches for obesity and metabolic disorders.

Upregulation of Mineralocorticoid Receptor in the Hypothalamus Associated with a High Anxiety-like Level in Apolipoprotein E4 Transgenic Mice.

Anxiety symptoms occur in a large portion of Alzheimer's disease (AD) patients. ApolipoproteinE-4 (ApoE ε4 allele), a risk factor for AD, has been recognized as an important contributor to psychiatric disorders. In the present study, we aimed to investigate the corticosterone level in relation to anxiety-like behavior changes in transgenic male mice with different glial fibrillary acidic protein (GFAP)-ApoE isoforms. GFAP-ApoE4 transgenic mice aged 3 months showed higher anxiety-like behavior in open field, light-dark box and elevated plus maze tasks compared with that of age-matched GFAP-ApoE3 mice. However, corticotropin releasing factor levels in the hypothalamus and plasma corticosterone secretion were similar in GFAP-ApoE3 and GFAP-ApoE4 transgenic male mice. Additionally, increased expression of the mineralocorticoid receptor (MR) and unchanged expression of the glucocorticoid receptor were observed in the hypothalamus of GFAP-ApoE4 mice. However, no significant differences were found in the expression levels of the MR in GFAP-ApoE3 and GFAP-ApoE4 mice at postnatal day 2. In conclusion, we found that MR upregulation rather than corticosterone level changes in the early stage of adulthood was associated with the higher anxiety-like level measured in GFAP-ApoE4 mice.

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.