Search

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca2+ imaging experiments in neurons, they are less useful for analyzing Ca2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)—a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.

In vitro studies performed on brain slices demonstrate that the potassium channel blocker 4-aminopyridine (4AP, 50 µM) discloses electrographic seizure activity and interictal discharges. These epileptiform patterns have been further analyzed here in a isolated whole guinea pig brain in vitro by using field potential recordings in olfactory and limbic structures. In 8 of 13 experiments runs of fast oscillatory activity (fast runs, FRs) in the piriform cortex (PC) propagated to the lateral entorhinal cortex (EC), hippocampus and occasionally to the medial EC. Early and late FRs were asynchronous in the hemispheres showed different duration [1.78 ± 0.51 and 27.95 ± 4.55 (SD) s, respectively], frequency of occurrence (1.82 ± 0.49 and 34.16 ± 6.03 s) and frequency content (20–40 vs. 40–60 Hz). Preictal spikes independent from the FRs appeared in the hippocampus/EC and developed into ictal-like discharges that did not propagate to the PC. Ictal-like activity consisted of fast activity with onset either in the hippocampus (n = 6) or in the mEC (n = 2), followed by irregular spiking and sequences of diffusely synchronous bursts. Perfusion of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid (100 µM) did not prevent FRs, increased the duration of limbic ictal-like discharges and favored their propagation to olfactory structures. The AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (50 µM) blocked ictal-like events and reduced FRs. In conclusion, 4AP-induced epileptiform activities are asynchronous and independent in olfactory and hippocampal-entorhinal regions. Epileptiform discharges in the isolated guinea pig brain show different pharmacological properties compared with rodent in vitro slices. [ABSTRACT FROM AUTHOR]

The olfactory tubercle (OT) is a cortical component of the olfactory system involved in reward mechanisms of drug abuse. This region covers an extensive part of the rostral ventral cerebrum and is relatively poorly studied. The intrinsic network interactions evoked by olfactory input are analyzed in the OT of the in vitro isolated guinea pig brain by means of field potential analysis and optical imaging of voltage-sensitive signals. Stimulation of the lateral olfactory tract induces a monosynaptic response that progressively decreases in amplitude from lateral to medial. The monosynaptic input induces a disynaptic response that is proportionally larger in the medial portion of the OT. Direct stimulation of the piriform cortex and subsequent lesion of this pathway showed the existence of an associative disynaptic projection from the anterior part of the piriform cortex to the lateral part of the OT that integrates with the component mediated by the local intra-OT collaterals. Optical and electrophysiological recordings of the signals evoked by stimulation of the olfactory tract during arterial perfusion with the voltage-sensitive dye di-2-ANEPEQ confirmed the pattern of distribution of the mono and disynaptic responses in the OT. Finally, current source density analysis of laminar profiles recorded with 16-channel silicon probes confirmed that the monosynaptic and disynaptic potentials localize in the most superficial and the deep portions of the plexiform layer I, as suggested by previous reports. This study sets the standard for further analysis of the modulation of network properties in this largely unexplored brain region. [ABSTRACT FROM AUTHOR]