80µm fixed brain slices (5100mz); Calcium Imaging & Patch study in 300µm brain slices (7000smz-2)

Abstract

(from Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo, Dorgans et al, 2022)

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Adeno-associated viral (AAV) vectors, used as vehicles for gene transfer into the brain, are a versatile and powerful tool of modern neuroscience that allow identifying specific neuronal populations, monitoring and modulating their activity. For consistent and reproducible results, the AAV vectors must be engineered so that they reliably and accurately target cell populations. Furthermore, transgene expression must be adjusted to sufficient and safe levels compatible with the physiology of studied cells. We undertook the effort to identify and validate an AAV vector that could be utilized for researching the inferior olivary (IO) nucleus, a structure gating critical timing-related signals to the cerebellum. By means of systematic construct generation and quantitative expression profiling, we succeeded in creating a viral tool for specific and strong transfection of the IO neurons without adverse effects on their physiology. The potential of these tools is demonstrated by expressing the calcium sensor GCaMP6s in adult mouse IO neurons. We could monitor subtle calcium fluctuations underlying two signatures of intrinsic IO activity: the subthreshold oscillations (STOs) and the variable-duration action potential waveforms both in-vitro and in-vivo. Further, we show that the expression levels of GCaMP6s allowing such recordings are compatible with the delicate calcium-based dynamics of IO neurons, inviting future work into the network dynamics of the olivo-cerebellar system in behaving animals.

Methods

Anatomical Study of Transgene Expression in IOn: Virus Preparation and Injection

For anatomical quantification of transfection results, animals were intracardially perfused 30 days after injection using at least 50 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). After fixation, the brain was extracted and immersed for 6 h in the same PFA solution before washing in PBS. Coronal brainstem and sagittal cerebellar sections (80 μm thick) were cut with a vibratome (5100MZ-plus; Campden Instruments, Loughborough, UK) equipped with ceramic blades (38 x 7 x 0.5 mm ceramic blades, model 7550-1-C, Campden Instruments, Loughborough, UK) and mounted on objective glass with Vectashield (H-1200, VectorLabs, CA) mounting medium and # 1.5 coverslip glass (Harvard Apparatus, MA).

Preparation of Acute IO Slices for in-vitro Calcium Imaging and Patch-Clamp Experiments

For in-vitro calcium imaging, animals that had been injected with the viral constructs to express GCaMP6s were used after 2 weeks of transfection time. The acute IO slice preparation has been previously described in detail. In brief, the animals were anesthetized with lethal dose of isoflurane and decapitated. Brainstem is extracted in warm standard physiological solution (SPS; composition: 126 mmol NaCl, 10 mmol glucose and 26 mmol NaHCO3, 3.4 ml KCl, 1.2 ml KH2PO4, then 1.3 ml MgSO4 (1 M), 2 mL CaCl2 (1 M); pH to 7.2–7.3; 34°C). 300 um thick coronal brainstem slices were cut with a vibrating microtome (7,000 smz-2, Campden Instruments, UK) equipped with ceramic blades (model 7550-1-C, Campden Instruments, UK) at low slicing speed (0.01 mm/s). After at least 1 h recovery period, slices were transferred to a submerged-type recording chamber, continuously perfused with warm (34°C) SPS. Slices were viewed with an upright microscope (BXW51, Olympus, JP) with 5x (NA:0.1, MPLN5X, Olympus, Japan) and 60x (NA:1.0, LUMPLFLN60XW, Olympus, Japan) objectives.