Neuronal and Astroglial Correlates Underlying Spatiotemporal Intrinsic Optical Signal in the Rat Hippocampal Slice

Abstract

Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na+ channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg2+-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K+/Cl− cotransporter KCC2, but not for glial Na+/K+/Cl− cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl− and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future.

Activity‐dependent astrocyte swelling is mediated by pH‐regulating mechanisms

“…NKCC1 via its ability to translocate water (Zeuthen & MacAulay, ) and Kir4.1 via an association with AQP4 (Nagelhus et al, ). Experimental evidence, however, illustrated that NKCC1 was not involved in stimulus‐induced ECS shrinkage in rat hippocampal brain slices (Larsen et al, ; Pál et al, ), which aligned well with the negligible NKCC1 expression in astrocytes in vivo (Plotkin et al, ; Zhang et al, ), despite its robust expression in cultured astrocytes (Larsen et al, ; Su, Haworth, Dempsey, & Sun, ; Walz, ). Both genetic ablation (Haj‐Yasein et al, ) and pharmacological experiments (Larsen et al, ) revealed that activity‐dependent glial swelling occurred independently of Kir4.1‐mediated spatial buffering, thus ruling out its contribution to this phenomenon.…”

“…Stimulus‐induced extracellular space shrinkage has been assigned to cellular swelling of the glial compartment (Connors et al, ; MacVicar et al, ; Ransom et al, ), as was additionally illustrated in slices bathed in high [K + ] o by direct visualization of the fluorescent astrocytes (Florence, Baillie, & Mulligan, ). Nevertheless, swelling of the neurons may well constitute a portion of this event (Pál et al, ), as evident during cortical spreading depolarization (Steffensen, Sword, Croom, Kirov, & MacAulay, ). Historically, the activity‐induced glial cell swelling has been inferred to occur in direct relation to the molecular machinery underlying K + clearance in the extracellular space, partly based on results obtained in cultured astrocytes (Hertz et al, ; Kofuji & Newman, ; Nagelhus et al, ; Su, Kintner, Flagella, Shull, & Sun, ; Tas, Massa, Kress, & Koschel, ; Walz, ).…”

“…The network activity and resultant rise in extracellular [K + ] occurs in parallel to a shrinkage of the ECS, a phenomenon attributed to swelling of adjacent cells (Connors, Ransom, Kunis, & Gutnick, ; Dietzel, Heinemann, Hofmeier, & Lux, ; Ransom, Yamate, & Connors, ). The cell swelling has been assigned to astrocytic structures as it persists in enucleated optic nerve (MacVicar, Feighan, Brown, & Ransom, ) and is absent in optic nerve of young rats at a developmental age prior to maturation of glial cells (Ransom et al, ), although a contribution from the neuronal compartment cannot be excluded (Pál, Nyitrai, Kardos, & Héja, ). However, the molecular mechanisms underlying the activity‐dependent glia cell swelling remain unresolved: As the ECS shrinkage parallels the stimulus‐induced [K + ] o transients, K + ‐transporting mechanisms such as the Na + /K + /2Cl − cotransporter type 1 (NKCC1) and the inwardly rectifying K + channel Kir4.1 were proposed as molecular candidates underlying the glial cell swelling (MacAulay & Zeuthen, ; Nagelhus, Mathiisen, & Ottersen, ).…”

Optical imaging of cortical networks via intracortical microstimulation

“…Consequently there is a local increase in deoxyhemoglobin in the microvasculature followed by a rebound in the amount of oxyhemoglobin and increased blood flow. Multiple lines of research are actively trying to determine the precise mechanisms leading to these series of events, such as the role of neurotransmitters (Choi et al 2006;Pál et al 2013;Radhakrishnan et al 2011), astrocytes (Gurden 2013;Winship et al 2007), and the types of neural activity (e.g., preor postsynaptic; Devor et al 2005) that lead to hemodynamic changes. In our use of intrinsic optical imaging to investigate the neural response to cortical microstimulation, it is likely that the signal we are measuring is a combination of the direct and indirect activation of neurons and astrocytes.…”

Brain active transmembrane water cycling measured by MR is associated with neuronal activity

“…However, simultaneous changes (e.g., in cell morphology and volume) likely occur for any perturbation of membrane water transport. Experiments with long‐term neuronal activity suppression (AP 5,o plus DNQX o , TTX o , and low [ K o + ]) showed significant cell volume reduction, which might be caused by the reduction in neuronal and glial cell swelling associated with neuronal activity . From Equation , k io (p) is inversely proportional to cell diameter (assuming the cell shape is little changed and P W (p) is constant).…”