Mesoscopic Mapping of Stimulus-Selective Response Plasticity in the Visual Pathways Modulated by the Cholinergic System.

The cholinergic potentiation of visual conditioning enhances visual acuity and discrimination of the trained stimulus. To determine if this also induces long-term plastic changes on cortical maps and connectivity in the visual cortex and higher associative areas, mesoscopic calcium imaging was performed in head-fixed awake GCaMP6s adult mice before and after conditioning. The conditioned stimulus (0.03 cpd, 30°, 100% contrast, 1 Hz-drifting gratings) was presented 10 min daily for a week. Saline or Donepezil (DPZ, 0.3 mg/kg, s.c.), a cholinesterase inhibitor that potentiates cholinergic transmission, were injected prior to each conditioning session and compared to a sham-conditioned group. Cortical maps of resting state and evoked response to the monocular presentation of conditioned or non-conditioned stimulus (30°, 50 and 75% contrast; 90°, 50, 75, and 100% contrast) were established. Amplitude, duration, and latency of the peak response, as well as size of activation were measured in the primary visual cortex (V1), secondary visual areas (AL, A, AM, PM, LM, RL), retrosplenial cortex (RSC), and higher cortical areas. Visual stimulation increased calcium signaling in all primary and secondary visual areas, the RSC, but no other cortices. There were no significant effects of sham-conditioning or conditioning alone, but DPZ treatment during conditioning significantly decreased the integrated neuronal activity of superficial layers evoked by the conditioned stimulus in V1, AL, PM, and LM. The activity of downstream cortical areas was not changed. The size of the activated area was decreased in V1 and PM, and the signal-to-noise ratio was decreased in AL and PM. Interestingly, signal correlation was seen only between V1, the ventral visual pathway, and the RSC, and was decreased by DPZ administration. The resting state activity was slightly correlated and rarely affected by treatments, except between binocular and monocular V1 in both hemispheres. In conclusion, cholinergic potentiation of visual conditioning induced change in visual processing in the superficial cortical layers. This effect might be a key mechanism in the establishment of the fine cortical tuning in response to the conditioned visual stimulus.

Cholinergic potentiation of visual perception and vision restoration in rodents and humans.

BACKGROUND: The cholinergic system is a potent neuromodulator system that plays a critical role in cortical plasticity, attention, and learning. Recently, it was found that boosting this system during perceptual learning robustly enhances sensory perception in rodents. In particular, pairing cholinergic activation with visual stimulation increases neuronal responses, cue detection ability, and long-term facilitation in the primary visual cortex. The mechanisms of cholinergic enhancement are closely linked to attentional processes, long-term potentiation, and modulation of the excitatory/inhibitory balance. Some studies currently examine this effect in humans. OBJECTIVE: The present article reviews the research from our laboratory, examining whether potentiating the central cholinergic system could help visual perception and restoration. METHODS: Electrophysiological or pharmacological enhancement of the cholinergic system are administered during a visual training. Electrophysiological responses and perceptual learning performance are investigated before and after the training in rats and humans. This approach's ability to restore visual capacities following a visual deficit induced by a partial optic nerve crush is also investigated in rats. RESULTS: The coupling of visual training to cholinergic stimulation improved visual discrimination and visual acuity in rats, and improved residual vision after a deficit. These changes were due to muscarinic and nicotinic transmissions and were associated with a functional improvement of evoked potentials. In humans, potentiation of cholinergic transmission with 5 mg of donepezil showed improved learning and ocular dominance plasticity, although this treatment was ineffective in augmenting the perceptual threshold and electroencephalography. CONCLUSIONS: Potential therapeutic outcomes ought to facilitate vision restoration using commercially available cholinergic agents combined with visual stimulation in order to prevent irreversible vision loss in patients. This approach has the potential to help a large population of visually impaired individuals.

Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification Coupled to RNA Sequencing.

Small regulatory RNAs (sRNAs) are ubiquitous regulatory molecules expressed in living cells. In prokaryotes, sRNAs usually bind to target mRNAs to either promote their degradation or interfere with translation initiation. Because a single sRNA can regulate a considerable number of target mRNAs, we seek to identify those targets rapidly and reliably. Here, we present a robust method based on the co-purification of target mRNAs bound to MS2-tagged sRNAs expressed in vivo. After purification of the tagged-sRNA, we use RNAseq to determine the identity of all RNA interacting partners and their enrichment level. We describe how to analyze the RNAseq data through the Galaxy Project Platform bioinformatics tools to identify new mRNA targets. This technique is applicable to most sRNAs of E. coli and Salmonella.

Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli.

Small RNAs are key components of complex regulatory networks. These molecules can integrate multiple cellular signals to control specific target mRNAs. The recent development of high-throughput methods tremendously helped to characterize the full targetome of sRNAs. Using MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we reveal the targetomes of two sRNAs, CyaR and RprA. Interestingly, both CyaR and RprA interact with the 5'-UTR of hdeD mRNA, which encodes an acid-resistance membrane protein. We demonstrate that CyaR classically binds to the RBS of hdeD, interfering with translational initiation. We identified an A/U-rich motif on hdeD, which is bound by the RNA chaperone Hfq. Our results indicate that binding of this motif by Hfq is required for CyaR-induced degradation of hdeD mRNA. Additional data suggest that two molecules of RprA must bind the 5'-UTR of hdeD to block translation initiation. Surprisingly, while both CyaR and RprA sRNAs bind to the same motif on hdeD mRNA, RprA solely acts at the translational level, leaving the target RNA intact. By interchanging the seed region of CyaR and RprA sRNAs, we also swap their regulatory behavior. These results suggest that slight changes in the seed region could modulate the regulation of target mRNAs.