Inspiratory Off-Switch Mediated by Optogenetic Activation of Inhibitory Neurons in the preBötzinger Complex In Vivo.

The role of inhibitory neurons in the respiratory network is a matter of ongoing debate. Conflicting and contradicting results are manifold and the question whether inhibitory neurons are essential for the generation of the respiratory rhythm as such is controversial. Inhibitory neurons are required in pulmonary reflexes for adapting the activity of the central respiratory network to the status of the lung and it is hypothesized that glycinergic neurons mediate the inspiratory off-switch. Over the years, optogenetic tools have been developed that allow for cell-specific activation of subsets of neurons in vitro and in vivo. In this study, we aimed to identify the effect of activation of inhibitory neurons in vivo. Here, we used a conditional transgenic mouse line that expresses Channelrhodopsin 2 in inhibitory neurons. A 200 µm multimode optical fiber ferrule was implanted in adult mice using stereotaxic surgery, allowing us to stimulate inhibitory, respiratory neurons within the core excitatory network in the preBötzinger complex of the ventrolateral medulla. We show that, in anesthetized mice, activation of inhibitory neurons by blue light (470 nm) continuously or with stimulation frequencies above 10 Hz results in a significant reduction of the respiratory rate, in some cases leading to complete cessation of breathing. However, a lower stimulation frequency (4-5 Hz) could induce a significant increase in the respiratory rate. This phenomenon can be explained by the resetting of the respiratory cycle, since stimulation during inspiration shortened the associated breath and thereby increased the respiratory rate, while stimulation during the expiratory interval reduced the respiratory rate. Taken together, these results support the concept that activation of inhibitory neurons mediates phase-switching by inhibiting excitatory rhythmogenic neurons in the preBötzinger complex.

Evaluation of a mechanical lung model to test small animal whole body plethysmography.

Whole-body plethysmography (WBP) is an established method to determine physiological parameters and pathophysiological alteration of breathing in animals and animal models of a variety of diseases. Although frequently used, there is ongoing debate about what exactly is measured by whole-body-plethysmography and how reliable the data derived from this method are. Here, we designed an artificial lung model that enables a thorough evaluation of different predictions about and around whole-body plethysmography. Using our lung model, we confirmed that during WBP two components contribute to the pressure changes detected in the chamber: (1) the increase in the pressure due to heating and moistening of the air during inspiration, termed conditioning; (2) changes in the chamber pressure that depend on airway resistance. Both components overlap and contribute to the temporal pressure-profile measured in the chamber or across the wall of the chamber, respectively. Our data showed that a precise measurement of the breathing volume appears to be hindered by at least two factors: (1) the unknown relative contribution of each of these two components; (2) not only the air in the inspired volume is conditioned during inspiration, but also air within the residual volume and dead space that is recruited during inspiration. Moreover, our data suggest that the expiratory negative pressure peak that is used to determine the enhanced pause (Penh) parameter is not a measure for airway resistance as such but rather a consequence of the animal's response to the airway resistance, using forced or active expiration to overcome the resistance by a higher thoracic pressure.

Limitations of Sulforhodamine 101 for Brain Imaging.

Since 2004, the red fluorescent dye Sulforhodamine 101 (SR101) has been boosting the functional analysis of astrocytes in a functional environment in an unprecedented way. However, two major limitations have been challenging the usefulness of this tool for cellular imaging: (i) SR101 is not as specific for astrocytes as previously reported; and (ii) discoveries of severe excitatory side effects of SR101 are bearing the risk of unwanted alteration of the system of interest. In this article, we summarize the current knowledge about SR101-labeling protocols and discuss the problems that arise from varying of the staining protocols. Furthermore, we provide a testable hypothesis for the observed hyper-excitability that can be observed when using SR101.

Unspecific labelling of oligodendrocytes by sulforhodamine 101 depends on astrocytic uptake via the thyroid hormone transporter OATP1C1 (SLCO1C1).

The red fluorescent dye Sulforhodamine 101 (SR101) is often used as a marker for astrocytes, although variations of the staining protocol have been shown to influence the preferentially labeled cell type. Here we analyzed SR101-labeling of oligodendrocytes in the hippocampal slices preparation of PLP-EGFP mice. Using different staining protocols, we found robust SR101-labeled oligodendrocytes in the CA1 stratum radiatum of the hippocampus. Application of L-thyroxin, which is known to block SR101 transport into astrocytes via competitive inhibition of the multi-specific OATP1C1 (SLCO1C1) transporter, significantly reduced oligodendrocyte labeling. Since OATP1C1 is not expressed in oligodendrocytes, we conclude that oligodendrocyte labeling with SR101 requires SR101-uptake by astrocytes, which then diffuses to oligodendrocytes via heterotypic gap junctions of the pan-glial network. In summary, unequivocal identification of a particular cell type is not possible by SR101 only, hence caution is recommended when using SR101 in future studies.