Identification of the Pre-Botzinger Complex Inspiratory Center in Calibrated “Sandwich” Slices from Newborn Mice with Fluorescent Dbx1 Interneurons

Inspiratory active pre‐Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow‐up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1‐derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0–4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging‐based studies, as well as the application of other Cre‐dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory‐related oscillations of preBötC Dbx1‐derived neurons and thus breathing

Genetically Encoded Glutamate Indicators with Altered Color and Topology

Glutamate is one of the 20 common amino acids and of utmost importance for chemically mediated synaptic transmission in nervous systems. To expand the color palette of genetically encoded indicators for glutamate, we used protein engineering to develop a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1). Manipulating the topology of R-iGluSnFR1, and a previously reported green fluorescent indicator, led to the development of noncircularly permutated (ncp) variants. R- and R^ncp-iGluSnFR1 display glutamate affinities of 11 μM and 0.9 μM, respectively. We demonstrate that these glutamate indicators are functional when targeted to the surface of HEK-293 cells. Furthermore, we show that G^ncp-iGluSnFR enabled reliable visualization of extrasynaptic glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR can reliably resolve action potential-evoked glutamate transients by electrical field stimuli in cultures of dissociated hippocampal neurons

Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem

The pre-Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstem–spinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K+] and [Ca2+] (both of which determine neuronal excitability) is not clear. We report here that inspiratory-related rhythms in newborn rat slices and brainstem–spinal cords with defined boundaries were basically similar in physiological Ca2+ (1.2 mm) and K+ (3 mm). Hypoglossal nerve rhythm was 1 : 1-coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC-related slice rhythms were inhibited in 1.5 mm Ca2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mm Ca2+. Ca2+ depression of slice rhythms was antagonized by raising superfusate K+ to 8–10 mm. This strong extracellular Ca2+/K+ antagonism of inspiratory (motor) rhythms was also revealed in brainstem–spinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K+, respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscle