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Dissecting neural connectivity patterns within local brain regions is an essential step to understanding the function of the brain.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

Summary Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Muller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson’s disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.

U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), a widely used mitogen-activated protein kinase kinase (MEK) inhibitor, was found to accelerate voltage-gated K+ channel (KV) inactivation in heterologous cells expressing several types of KV. The goal of this study was to examine whether U0126 at a concentration thought to specifically inhibit MEK signaling also inhibits KV in native neurons of primary cultures or brain slices. U0126 caused a dose-dependent inhibition of both the transient (IA) and sustained (IDR) components of K+ currents in hippocampal neurons. U0126 also exhibited much higher potency on the IA and IDR than the classical KV blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Consistent with its inhibitory effect on KV, U0126 broadened action potential duration, profoundly affected the repolarizing phase, and dramatically reduced firing frequency in response to current pulse injections. Despite the potent and reversible action of U0126 on Kv channels, PD98059, a structurally-unrelated MEK inhibitor, did not induce such an effect, suggesting U0126 may act independently of MEK inhibition. Together, these results raise cautions for using U0126 as a specific inhibitor for studying MEK signaling in neurons; on the other hand, further studies on the blocking mechanisms of U0126 as a potent inhibitor of KV may provide useful insights into the structure-function relationship of KV in general.

Determining the degree of synapse formation and elimination is essential for understanding the structural basis of brain plasticity and pathology. We show that in vivo imaging of dendritic spine dynamics through an open-skull glass window, but not a thinned-skull window, is associated with high spine turnover and substantial glial activation during the first month after surgery. These findings help to explain existing discrepancies in the degree of dendritic spine plasticity observed in the mature cortex.

A fundamental feature of the mammalian neocortex is its columnar organization. In the visual cortex, functional columns consisting of neurons with similar orientation preferences have been characterized extensively, but how these columns are constructed during development remains unclear. The radial unit hypothesis posits that the ontogenetic columns formed by clonally related neurons migrating along the same radial glial fibre during corticogenesis provide the basis for functional columns in adult neocortex. However, a direct correspondence between the ontogenetic and functional columns has not been demonstrated. Here we show that, despite the lack of a discernible orientation map in mouse visual cortex, sister neurons in the same radial clone exhibit similar orientation preferences. Using a retroviral vector encoding green fluorescent protein to label radial clones of excitatory neurons, and in vivo two-photon calcium imaging to measure neuronal response properties, we found that sister neurons preferred similar orientations whereas nearby non-sister neurons showed no such relationship. Interestingly, disruption of gap junction coupling by viral expression of a dominant-negative mutant of Cx26 (also known as Gjb2) or by daily administration of a gap junction blocker, carbenoxolone, during the first postnatal week greatly diminished the functional similarity between sister neurons, suggesting that the maturation of ontogenetic into functional columns requires intercellular communication through gap junctions. Together with the recent finding of preferential excitatory connections among sister neurons, our results support the radial unit hypothesis and unify the ontogenetic and functional columns in the visual cortex.

To study the effect of stigma maydis polysaccharide (SMPS) on gastrointestinal movement.Taking charcoal as the indicator and taking ratio of charcoal movement, beginning time of black excretion and stool amount as the index to observe the effect of SMPS on intestinal movement in mice. Taking emthylorange as the indicator and taking the ratio of residual rate of methylorange as the index to observe the effect of SMPS on gastric emptying in mice. Taking methylene blue as the indicator and taking the time of gastric emptying and movement speed of intestinal content as the index to observe the effect of SMPS on gastrointestinal movement in rats. Observing the changes of cholecystokinin (CCK) level in plasm in rats.Compared with control, the ratio of charcoal movement increased in mice (P0.01). The beginning time of black excretion shortened and the stool amount increased in mice (P0.01). The ratio of residual rate of methylorange increased in mice (P0. 01). The time of gastric emptying prolonged in rats (P0.01). The movement speed of intestinal content in rats accelerated (P0.01). CCK level in plasm increased in rats (P0.05).Effects of stigma maydis polysaccharide on gastrointestinal movement are probably related to the increasing of CCK level in plasm.

To study the effects of Rheum tanguticum polysaccharide(-1) (RTP(-1)) on ulcerative colitis in rats induced by 2, 4, 6-trinitrophene sulphonic acid (TNBS) and their possible mechanism.RTP1 (200 mg.kg(-1), i.g.) extracted from Rheum tanguticum Maxim. ex Regel was administrated to rats with colitis induced by TNBS for 5 d, 7 d, 10 d and 14 d, respectively. The effects of RTP1 and dexamethasone (DX, 0.2 mg.kg(-1), i.g.) were contrastively investigated. The MPO level and SOD activity were determined by chromatometry. The expansion and protein expression of CD4+T lymphocytes isolated from colon mucosae and mesenteric lymph nodes of colitis rats were performed by immunohistochemical analysis and Western-blot methods.Treatments of RTP1 (200 mg.kg(-1), i.g.) significantly reduced diarrhea, mortality, colon mass, ulcer areas and MPO level in colon mucosae on days 5, 7, 10 and 14 (5.2+/-1.4, 5.4+/-0.7, 5.2+/-1.8, P0.05. 3.4+/-0.8, P0.01. 16.1+/-12.1, P0.01. 31.8+/-8.6, 17.7+/-5.3, 12.7+/-4.1, P0.05). The effects of RTP1 were similar to those noted above in DX group, but there were no immunosuppressive effects of DX in RTP(-1) group, such as body mass loss, thymus and spleen atrophy. The decreased number and down-regulated protein levels of CD4+T cells isolated from the colon of colitis rats treated with RTP1 were found.RTP1 shows significantly protective effects but lower side effects on rats with colitis induced by TNBS. The mechanism may be due to the resistance to over expansion of CD4.