In this protocol, the authors describe the preparation and maintenance of rat organotypic cultures (cortico-striatal) for AAV infection and imaging of the extracellular matrix.

In this protocol we describe the preparation and maintenance of rat organotypic cultures from parasagittal brain slices. We use a 13º slicing angle and a customized cocktail of small molecules and growth factors, to maximize the integrity of the nigrostriatal pathway and ensure the survival of dopaminergic neurons, respectively. The slices maintain the basic cytoarchitecture of the brain, including glia and the extracellular matrix (not including vessels nor immune system). The cultures can be used to study dopaminergic degeneration, cell-cell and cell-matrix interactions and are particularly suitable for AAV-mediated overexpression of transgenes, time-lapse live imaging and longitudinal studies.

Quantification of area and optical density of intracellular neuromelanin with Image J.

Quantification of area and optical density of intracellular neuromelanin with TruAI in H&E stained sections.

Protocol for preparing post-mortem tissue for intracellular neuromelanin quantification in H&E-stained sections.

Quantification of area and optical density of intracellular neuromelanin with TruAI.

This protocol describes preparation of organotypic hemisphere cultures prepared from mouse brain and cultured as rollerdrum cultures on glass coverslips.

Protocol for preparing post-mortem tissue for intracellular NM quantification.

Protocol for Sholl analysis by Neurolucida software.

This protocol describes an automatizable pipeline for cell counting based on fluorescent stainings with the help of CellPose.

This protocol covers chronic optrode construction, implantation, and recording in the Locus coeruleus (LC). It begins with building the optrode, involving assembly of a plastic housing, screw, thumbnut, and electrode interface board (EIB-32), followed by attaching electrodes and optic fibers to the EIB-32. The electrodes are then glued and trimmed, ensuring protection from animal-related damage. Implantation involves anesthesia, craniotomy, and careful electrode positioning. A gradual approach is recommended for electrode insertion to minimize tissue damage. The optrode is secured with cement and vaseline for stability. For recording from the LC, an OpenEphys system with omnetics headstage and acquisition software is used. Animals undergo 30-minute sessions with opto-tagging via red or blue light pulses. The setup ensures precise data acquisition and identification of neuron responses.

The Fontana-Masson Staining Protocol is a detailed method for staining free-floating sections (20-50 microns) to detect melanin and other pigments in tissue samples. The procedure involves preparing a silver nitrate working solution, treating sections with triton, and sequential incubation in pre-heated silver nitrate solution, gold chloride, and thiosulfate solutions, with thorough washing steps between each incubation. The process concludes with sections being placed in PBS for mounting and counterstaining. This protocol is critical for histological studies in biology, particularly for pigment analysis in tissues.

The Live Mouse Tracker (LMT) system is engineered for long-term, automated tracking and behavioral analysis of mice, integrating machine learning, computer vision, and RFID technology for simultaneous monitoring of multiple subjects. Specifically designed for the research community, LMT offers a Python-based framework for in-depth data analysis and is adept at facilitating studies involving mouse vocalizations. This cost-effective, DIY system is compatible with Windows OS and is tailored for tracking mouse behavior. Currently employed in our studies, LMT is pivotal in observing behavioral shifts across various domains such as arousal-resting, social interaction, and motor functions in a Neuromelanin-accumulating early-stage Parkinson’s Disease model utilizing the tyrosinase approach.