Gliotoxin‐induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead‐space microdomains

Abstract

One of the hallmarks of numerous life-threatening and debilitating brain diseases is cellular swelling that negatively impacts extracellular space (ECS) structure. The ECS structure is determined by two macroscopic parameters, namely tortuosity (λ) and volume fraction (α). Tortuosity represents hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium. Volume fraction is the proportion of tissue volume occupied by the ECS. From a clinical perspective, it is essential to recognize which factors determine the ECS parameters and how these factors change in brain diseases. Previous studies demonstrated that dead-space (DS) microdomains increased λ during ischemia and hypotonic stress, as these pocket-like structures transiently trapped diffusing molecules. We hypothesize that astrocytes play a key role in the formation of DS microdomains because their thin processes have concave shapes that may elongate as astrocytes swell in these pathologies. Here we selectively swelled astrocytes in the somatosensory neocortex of rat brain slices with a gliotoxin DL-α-Aminoadipic Acid (DL-AA), and we quantified the ECS parameters using Integrative Optical Imaging (IOI) and Real-Time Iontophoretic (RTI) diffusion methods. We found that α decreased and λ increased during DL-AA application. During recovery, α was restored whereas λ remained elevated. Increase in λ during astrocytic swelling and recovery is consistent with the formation of DS microdomains. Our data attribute to the astrocytes an important role in determining the ECS parameters, and indicate that extracellular diffusion can be improved not only by reducing the swelling but also by disrupting the DS microdomains.

ATP and astrocytes play a prominent role in the control of the respiratory pattern generator in the lamprey

“…; Sherpa et al . ). The application time and the latency of the effects were similar to those described in previous reports using not only AAA, but also other gliotoxins in different preparations, thus implying that a relatively long latency is necessary to impair astrocytes (Hülsmann et al .…”

“…; Sherpa et al . ). As a rule, each drug was allowed to perfuse the brainstem for 30 min, with the exception of AAA, which was applied to the bath for ∼60 min or more (see Results) owing to its relatively slowly developing action (Baudoux & Parker, ; Sherpa et al .…”

“…As a rule, each drug was allowed to perfuse the brainstem for 30 min, with the exception of AAA, which was applied to the bath for ∼60 min or more (see Results) owing to its relatively slowly developing action (Baudoux & Parker, ; Sherpa et al . ). After completion of each drug challenge, the preparation was allowed to recover by perfusing it with the control solution.…”

“…Experiments were carried out on isolated brainstems of adult lampreys by using bath application or microinjections of drugs into the pTRG to address the following issues: (1) the presence and type of purinergic receptors at the pTRG level by investigating the effects of ATP and those of specific agonists and antagonists on respiratory activity; (2) the function of astrocytes in the control of the respiratory pattern generator by the application of the gliotoxin l ‐2‐aminoadipic acid (AAA; Baudoux & Parker, ; Sherpa et al . ); (3) the involvement of astrocytes and ATP in the increase in respiratory activity induced by the acidification of the perfusing solution at constant ; (4) the immunohistochemical detection of astrocytes at the level of the pTRG under control conditions and after AAA application.…”

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

“…Prepare rodent brain slices with a thickness of 400 μm according to standard protocols 11,12 . Use the ACSF prepared in step 5.1 for the dissection and maintenance of brain slices.…”