ViNe-Seg: deep-learning-assisted segmentation of visible neurons and subsequent analysis embedded in a graphical user interface.

SUMMARY: Segmentation of neural somata is a crucial and usually the most time-consuming step in the analysis of optical functional imaging of neuronal microcircuits. In recent years, multiple auto-segmentation tools have been developed to improve the speed and consistency of the segmentation process, mostly, using deep learning approaches. Current segmentation tools, while advanced, still encounter challenges in producing accurate segmentation results, especially in datasets with a low signal-to-noise ratio. This has led to a reliance on manual segmentation techniques. However, manual methods, while customized to specific laboratory protocols, can introduce variability due to individual differences in interpretation, potentially affecting dataset consistency across studies. In response to this challenge, we present ViNe-Seg: a deep-learning-based semi-automatic segmentation tool that offers (i) detection of visible neurons, irrespective of their activity status; (ii) the ability to perform segmentation during an ongoing experiment; (iii) a user-friendly graphical interface that facilitates expert supervision, ensuring precise identification of Regions of Interest; (iv) an array of segmentation models with the option of training custom models and sharing them with the community; and (v) seamless integration of subsequent analysis steps. AVAILABILITY AND IMPLEMENTATION: ViNe-Seg code and documentation are publicly available at https://github.com/NiRuff/ViNe-Seg and can be installed from https://pypi.org/project/ViNeSeg/.

Pipeline for 2-photon all-optical physiology in mouse: From viral titration and optical window implantation to binarization of calcium transients.

2-photon all-optical physiology combines in vivo 2-photon calcium imaging and optogenetics, which enables both the read out and manipulation of neuronal microcircuits with single-cell resolution. Here, we describe a protocol for achieving optimized co-expression of calcium indicator and opsin. To enable longitudinal designs, we introduce a template for virus injection and chronic window implantation. We also highlight key aspects of performing 2-photon imaging and suggest an analysis algorithm for the binarization of putatively action-potential (AP)-related calcium transients. For complete details on the use and execution of this protocol, please refer to Fu et al. (2021).

Exploring two-photon optogenetics beyond 1100 nm for specific and effective all-optical physiology.

Two-photon (2-P) all-optical approaches combine in vivo 2-P calcium imaging and 2-P optogenetic modulations. Here, firstly, we combined in vivo juxtacellular recordings and GCaMP6f-based 2-P calcium imaging in mouse visual cortex to tune our detection algorithm towards a 100% specific identification of action potential-related calcium transients. Secondly, we minimized photostimulation artifacts by using extended-wavelength-spectrum laser sources for optogenetic stimulation. We achieved artifact-free all-optical experiments performing optogenetic stimulation from 1100 nm to 1300 nm. Thirdly, we determined the spectral range for maximizing efficacy until 1300 nm. The rate of evoked transients in GCaMP6f/C1V1-co-expressing cortical neurons peaked already at 1100 nm. By refining spike detection and defining 1100 nm as the optimal wavelength for artifact-free and effective GCaMP6f/C1V1-based all-optical physiology, we increased the translational value of these approaches, e.g., for the development of network-based therapies.

Cortex-wide BOLD fMRI activity reflects locally-recorded slow oscillation-associated calcium waves.

Spontaneous slow oscillation-associated slow wave activity represents an internally generated state which is characterized by alternations of network quiescence and stereotypical episodes of neuronal activity - slow wave events. However, it remains unclear which macroscopic signal is related to these active periods of the slow wave rhythm. We used optic fiber-based calcium recordings of local neural populations in cortex and thalamus to detect neurophysiologically defined slow calcium waves in isoflurane anesthetized rats. The individual slow wave events were used for an event-related analysis of simultaneously acquired whole-brain BOLD fMRI. We identified BOLD responses directly related to onsets of slow calcium waves, revealing a cortex-wide BOLD correlate: the entire cortex was engaged in this specific type of slow wave activity. These findings demonstrate a direct relation of defined neurophysiological events to a specific BOLD activity pattern and were confirmed for ongoing slow wave activity by independent component and seed-based analyses.